Thienopyridine compounds, their production and use

ABSTRACT

The compound of the present invention possesses excellent gonadotropin-releasing hormone antagonizing activity, and is useful for preventing or treating sex hormone-dependent diseases, e.g., sex hormone-dependent cancers (e.g., prostatic cancer, uterine cancer, breast cancer, pituitary tumor), prostatic hypertrophy, hysteromyoma, endometriosis, precocious puberty, amenorrhea syndrome, multilocular ovary syndrome, pimples etc. or as a pregnancy regulator (e.g., contraceptive), infertility remedy or menstruation regulator.

This application is a continuation of application Ser. No. 09/402,206now U.S. Pat. No. 6,262,267, filed Sep. 30, 1999 which was the NationalStage of International Patent Application Ser. No. PCT/JP99/03379, filedJun. 24, 1999.

TECHNICAL FIELD

The present invention relates to thieno [2,3-b ]pyridine derivativesexhibiting gonadotropin releasing hormone (GnRH) antagonizing activity,their production and use.

BACKGROUND ART

The secretion of hypophysial anterior lobe hormone is regulated by theperipheral hormone secreted by each target organ and thesecretion-promoting or secretion-suppressing hormone secreted by thehypothalamus, which is the center superior to the hypophysial anteriorlobe, and this group of hormones hereinafter generically referred to ashypothalamic hormone in this specification. To date, nine hypothalamichormones have been identified, for example, thyroid-stimulatinghormone-releasing hormone (TRH), and gonadotropin releasing hormone[GnRH, also known as luteinizing hormone releasing hormone (LH-RH)],etc. It is conjectured that these hypothalamic hormones exhibit theirhormone actions etc. via receptors assumed to be present in thehypophysial anterior lobe, and analyses of receptor genes specific tothese hormones, including humans, are ongoing. Antagonists or agoniststhat act specifically and selectively on these receptors would thereforeregulate the action of hypothalamic hormones and hence regulate thesecretion of hypophysial anterior lobe hormone. As a result, suchantagonists or agonists are expected to prevent or treat diseasesdepending on these hypophysial anterior lobe hormone.

Known compounds possessing GnRH-antagonizing activity includeGnRH-derived linear peptides (U.S. Pat. Nos. 5,140,009 and 5,171,835), acyclic hexapeptide derivative (JP-A-61-191698), a bicyclic peptidederivative [Journal of Medicinal Chemistry, Vol. 36, pp. 3265-3273(1993)], and so forth. Non-peptide compounds possessingGnRH-antagonizing activity include compounds described in WO 95/28405,WO 97/14697, WO 97/14682, WO 97/41126 and so forth.

Peptide compounds pose a large number of problems to be resolved withrespect to oral absorbability, dosage form, dose volume, drug stability,sustained action, metabolic stability etc. There is strong demand for anoral GnRH antagonist, especially one based on a non-peptide compound,that has excellent therapeutic effect on hormone-dependent cancers,e.g., prostatic cancer, endometriosis, precocious puberty etc., and thatdoes not show transient hypophysial-gonadotropic action (acute action).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagrammatic representation of the ameliorating effect ofsuppression of plasma LH concentrations in castrated monkeys after oraladministration of the compound of the following Example 1-2, in which .. . ▪. . . represents control (1) (vehicle), . . . ♦. . . representscontrol (2) (vehicle), . . . □. . . represents control (3) (vehicle),—Δ— represents compound (1)[the test animal administered the compound ofEx. No. 1-2]and —▴— represents compound (2) [the test animaladministered the compound of Ex. No. 1-2].

DISCLOSURE OF INVENTION

The present inventors produced various thienopyridine derivatives,investigated their actions, and found that some compounds possessexcellent GnRH-antagonizing activity. The inventors have conductedfurther investigation based on this finding, and developed the presentinvention.

Accordingly, the present invention relates to:

[1] a compound of the formula:

wherein R¹ represents a C₁₋₇ alkyl group which may be substituted, aC₃₋₇ cycloalkyl group which may be substituted, a C₁₋₆ alkoxyamino groupwhich may be substituted or a hydroxyamino group which may besubstituted; and

R² represents a C₁₋₇ alkyl group which may be substituted or a phenylgroup which may be substituted; when R¹ is an unsubstituted C₁₋₇ alkylgroup, then R² is a substituted C₁₋₇ alkyl group or a substituted phenylgroup, or a salt thereof [hereinafter sometimes referred to briefly ascompound (I)];

[2] a compound of the above (1) or a salt thereof, wherein R¹ is

(1) a C₁₋₇ alkyl group which may be substituted by 1 to 5 substituentsselected from the group consisting of (i) hydroxy, (ii) C₁₋₇ acyloxy,(iii) amino which may be substituted by 1 or 2 substituents selectedfrom the group consisting of C₁₋₆ alkoxy-carbonyl, benzyloxycarbonyl,C₁₋₃ acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iv) C₁₋₁₀ alkoxy whichmay be substituted by 1 to 3 substituents selected from the groupconsisting of C₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxy and (v) C₁₋₆alkoxy-carbonyl,

(2) a C₃₋₇ cycloalkyl group which may be substituted by 1 to 3substituents selected from the group consisting of (i) hydroxy, (ii)C₁₋₇ acyloxy, (iii) amino which may be substituted by 1 or 2substituents selected from the group consisting of C₁₋₆ alkoxy-carbonyl,benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iv)C₁₋₁₀ alkoxy which may be substituted by 1 to 3 substituents selectedfrom the group consisting of C₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxyand (v) C₁₋₆ alkoxy-carbonyl,

(3) a C₁₋₆ alkoxyamino group which may be substituted by 1 to 5substituents selected from the group consisting of (i) hydroxy, (ii)C₁₋₇ acyloxy, (iii) amino which may be substituted by 1 or 2substituents selected from the group consisting of C₁₋₆ alkoxy-carbonyl,benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iv)C₁₋₁₀ alkoxy which may be substituted by 1 to 3 substituents selectedfrom the group consisting of C₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxyand (v) C₁₋₆ alkoxy-carbonyl, or

(4) a hydroxyamino group which may be substituted by 1 or 2 substituentsselected from the group consisting of (i) C₁₋₇ acyloxy, (ii) amino whichmay be substituted by 1 or 2 substituents selected from the groupconsisting of C₁₋₆ alkoxy-carbonyl, benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃alkylsulfonyl and C₁₋₃ alkyl, (iii) C₁₋₁₀ alkoxy which my be substitutedby 1 to 3 substituents selected from the group consisting of C₃₋₇cycloalkyloxy-carbonyl and C₁₋₃ alkoxy and (iv) C₁₋₆ alkyl; and

R² is

(1) a C₁₋₇ alkyl group which may be substituted by 1 to 5 substituentsselected from the group consisting of (i) hydroxy, (ii) C₁₋₇ acyloxy,(iii) amino which may be substituted by 1 or 2 substituents selectedfrom the group consisting of C₁₋₆ alkoxy-carbonyl, benzyloxycarbonyl,C₁₋₃ acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iv) C₁₋₁₀ alkoxy whichmay be substituted by 1 to 3 substituents selected from the groupconsisting of C₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxy and (v) C₁₋₆alkoxy-carbonyl, or

(2) a phenyl group which may be substituted by 1 to 5 substituentsselected from the group consisting of halogen, C₁₋₃ alkyl and C₁₋₃alkoxy;

[3] a compound of the above (1) or a salt thereof, wherein R¹ is a C₁₋₇alkyl group which may be substituted or a C₃₋₇ cycloalkyl group whichmay be substituted;

[4] a compound of the above (1) or a salt thereof, wherein R¹ is asubstituted branched C₃₋₇ alkyl group or a substituted C₃₋₇ cycloalkylgroup;

[5] a compound of the above (1) or a salt thereof, wherein R¹ is a C₁₋₇alkyl group substituted by hydroxy or a C₃₋₇ cycloalkyl groupsubstituted by hydroxy;

[6] a compound of the above (1) or a salt thereof, wherein R¹ is asubstituted C₃₋₇ cycloalkyl group;

[7] a compound of the above (1) or a salt thereof, wherein R¹ is acyclopropyl group which may be substituted by hydroxy;

[8] a compound of the above (1) or a salt thereof, wherein R² is abranched C₃₋₇ alkyl group which may be substituted;

[9] a compound of the above (1) or a salt thereof, wherein R² is aphenyl group which may be substituted;

[10] a compound of the above (1) or a salt thereof, wherein R² is aphenyl group;

[11] a compound of the above (1) or a salt thereof, wherein R¹ is a C₃₋₇cycloalkyl group and R² is a C₁₋₆ alkyl group;

[12] a compound of the above (1) or a salt thereof, wherein R¹ is (1) aC₄ alkyl group substituted by 1 or 2 hydroxy, (2) a C₃₋₇ cycloalkylgroup substituted by hydroxy, or (3) a C₁₋₃ alkoxyamino group; and

R² is an isopropyl group or a phenyl group;

[13] a compound of the above (1) or a salt thereof, wherein R¹ is (1) aC₁₋₇ alkyl group which may be substituted by 1 or 2 substituentsselected from the group consisting of hydroxy, C₁₋₃ alkyl-carbonyloxy,amino, benzyloxycarbonylamino, C₁₋₃ alkoxy, C₁₋₃ alkoxy-C₁₋₃ alkoxy andC₁₋₃ alkoxy-carbonyl, (2) a C₃₋₇ cycloalkyl group which may besubstituted by a hydroxy or a C₁₋₃ alkyl-carbonyloxy, or (3) a C₁₋₃alkoxyamino group; and

R² is (1) an isopropyl group which may be substituted by a hydroxy or(2) a phenyl group;

[14]3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-cyclopropanecarbonylaminophenyl)-4-oxothieno[2,3-b]pyridineor a salt thereof;

[15]3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]-4-oxothieno[2,3-b]pyridineor a salt thereof;

[16]3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-[(1-hydroxycyclopropyl)carbonylaminolphenyl]-4-oxothieno[2,3-b]pyridineor a salt thereof;

[17]3-(N-benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridineor a salt thereof;

[18] a process f or producing a compound of the above [1] or a saltthereof, which comprises reacting a compound of the formula:

wherein R² represents a C₁₋₇ alkyl group which may be substituted or aphenyl group which may be substituted, or a salt thereof (hereinaftersometimes referred to briefly as compound (II)], with

i) a compound of the formula:

R^(1a)COOH

wherein R^(1a) represents a C₁₋₇ alkyl group which may be substituted ora C₃₋₇ cycloalkyl group which may be substituted, or a salt thereof or areactive derivative thereof; or

ii) carbonyldiimidazole, phosgene or a chloroformate, followed byreacting with a compound of the formula:

R^(1b)H

wherein R^(1b) represents a C₁₋₆ alkoxyamino group which may besubstituted or a hydroxyamino group which may be substituted, or a saltthereof;

[19] a process for producing a compound of the above [3] or a saltthereof, which comprises reacting compound (II) with a compound of theformula:

R^(1a)COOH

wherein R^(1a) represents a C₁₋₇ alkyl group which may be substituted ora C₃₋₇ cycloalkyl group which may be substituted, or a salt thereof or areactive derivative thereof;

[20] a pharmaceutical composition which comprises a compound of theabove (1) or a salt thereof;

[21] a pharmaceutical composition of the above (20) which is forantagonizing gonadotropin-releasing hormone;

[22] a pharmaceutical composition of the above (21) which is forpreventing or treating a sex hormone dependent disease;

[23] a method for antagonizing gonadotropin-releasing hormone in amammal in need thereof which comprises administering to said mammal aneffective amount of a compound of the above (1) or a salt thereof with apharmaceutically acceptable excipient, carrier or diluent;

[24] use of a compound of the above (1) or a salt thereof formanufacturing a pharmaceutical composition for antagonizinggonadotropin-releasing hormone, and so forth.

Each symbol in the above formulae is hereinafter described in moredetail.

The “C₁₋₇ alkyl group” of the “C₁₋₇ alkyl group which may besubstituted” for R¹ includes, for example, a straight-chain C₁₋₇ alkylgroup such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, etc.;a branched C₃₋₇ alkyl group such as isopropyl, isobutyl, sec-butyl,tert-butyl, isopentyl, neopentyl, etc., and so forth. Among others,preferred is a branched C₃₋₇ alkyl group. More preferred is isopropyl.

The “substituents” of the “C₁₋₇ alkyl group which may be substituted”for R¹ include, for example, (i) hydroxy, (ii) C₁₋₇ acyloxy (e.g., C₁₋₆alkyl-carbonyloxy such as acetoxy, propionyloxy, etc.; benzoyloxy etc.),(iii) amino which may be substituted by 1 or 2 substituents selectedfrom the group consisting of C₁₋₆ alkoxy-carbonyl (e.g.,methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, etc.),benzyloxycarbonyl, C₁₋₃ acyl (e.g., C₁₋₂ alkyl-carbonyl such as acetyl,propionyl, etc.), C₁₋₃ alkylsulfonyl (e.g., methanesulfonyl etc.) andC₁₋₃ alkyl (e.g., methyl, ethyl, etc.), etc. [e.g.; amino,methoxycarbonylamino, ethoxycarbonylamino,tert-butoxycarbonylbenzyloxycarbonylamino, acetylamino,methanesulfonylamino, methylamino, dimethylamino, etc.), (iv) C₁₋₁₀(preferably C₁₋₄) alkoxy which may be substituted by 1 to 3 substituentsselected from the group consisting of C₃₋₇ cycloalkyloxycarbonyl (e.g.,cyclohexyloxycarbonyloxy, etc.) and C₁₋₃ alkoxy(e.g., methoxy, ethoxy,etc.) [e.g.; methoxy, ethoxy, propoxy, tert-butoxy,cyclohexyloxycarbonyloxy-1-ethoxy, methoxymethoxy, ethoxymethoxy, etc.],(v) C₁₋₆ alkoxy-carbonyl (e.g., methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, etc.), and so forth. Among others, preferred ishydroxy.

The “C₁₋₇ alkyl group” may have 1 to 5, preferably 1 to 3 substituentsas mentioned above at possible positions and, when the number ofsubstituents is two or more, those substituents may be the same as ordifferent from one another.

The “C₃₋₇ cycloalkyl group” of the “C₃₋₇ cycloalkyl group which may besubstituted” for R¹ includes, for example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, etc. Among others, preferred iscyclopropyl.

The “substituents” of the “C₃₋₇ cycloalkyl group which may besubstituted” for R¹ are the same as those mentioned above for the“substituents” of the “C₁₋₇ alkyl group which may be substituted” forR¹. The number of substituents is 1 to 3. When the number ofsubstituents is two or more, those substituents maybe the same as ordifferent from one another.

The “C₁₋₆ alkoxyamino group” of the “C₁₋₆ alkoxyamino group which may besubstituted” for R¹ includes, for example, mono- or di-C₁₋₆ alkoxyamino(e.g., methoxyamino, ethoxyamino, dimethoxyamino, diethoxyamino,ethoxymethoxyamino, etc.), etc. Among others, preferred is mono-C₁₋₃alkoxyamino (e.g., methoxyamino, etc.).

The “substituents” of the “C₁₋₆ alkoxyamino group which may besubstituted” for R¹ and their number are the same as those mentionedabove for the “substituents” of the “C₁₋₇ alkyl group which may besubstituted” for R¹. When the number of substituents is two or more,those substituents may be the same as or different from one another. The“C₁₋₆ alkoxy” or the “nitrogen atom of an amino group” of the C₁₋₆alkoxyamino group may be substituted by the above “substituents”.

Such “C₁₋₆ alkoxyamino group which may be substituted” is exemplified bymethoxyamino, N-methyl-N-methoxyamino, N-ethyl-N-methoxyamino,ethoxyamino, dimethoxyamino, diethoxyamino, ethoxymethoxyamino, etc.Preferred is C₁₋₃ alkoxyamino, N-C₁₋₃ alkyl-N-C₁₋₃ alkoxyamino, etc.

The “substituents” of the “hydroxyamino group which may be substituted”for R¹ may be located on the “hydroxy group” of the hydroxyamino groupor the “nitrogen atom of an amino group” of the hydroxyamino group. Such“substituents” on the “hydroxy group” include, for example, (i) C₁₋₇acyloxy (e.g., C₁₋₆ alkyl-carbonyloxy such as acetoxy, propionyloxy,etc.; benzoyloxy etc.), (ii) amino which may be substituted by 1 or 2substituents selected from the group consisting of C₁₋₆ alkoxy-carbonyl(e.g., methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, etc.),benzyloxycarbonyl, C₁₋₃ acyl (e.g., C₁₋₂ alkyl-carbonyl such as acetyl,propionyl, etc.), C₁₋₃ alkylsulfonyl (e.g., methanesulfonyl etc.) andC₁₋₃ alkyl (e.g., methyl, ethyl, etc.), etc. [e.g.; amino,methoxycarbonylamino, ethoxycarbonylamino,tert-butoxycarbonylbenzyloxycarbonylamino, acetylamino,methanesulfonylamino, methylamino, dimethylamino, etc.], (iii) C₁₋₁₀(preferably C₁₋₄ ) alkoxy which maybe substituted by 1 to 3 substituentsselected from the group consisting of C₃₋₇ cycloalkyloxycarbonyl (e.g.,cyclohexyloxycarbonyloxy, etc.) and C₁₋₃ alkoxy (e.g., methoxy, ethoxy,etc.) [e.g.; methoxy, ethoxy, propoxy, tert-butoxy,cyclohexyloxycarbonyloxy-1-ethoxy, methoxymethoxy, ethoxymethoxy, etc.],and so forth. The number of substituents is 1 to 5, preferably 1 to 3.When the number of substituents is two or more, those substituents maybe the same as or different from one another. Such “substituents” on the“nitrogen atom of the amino group” include, for example, (1) each groupas described in the above (i) to (iii) and (2) C₁₋₆ alkyl (e.g., methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, neopentyl, hexyl, etc. The number of substituents is1 to 5, preferably 1 to 3. When the number of substituents is two ormore, those substituents may be the same as or different from oneanother.

Preferable examples of the “hydroxyamino group which may be substituted”include N-C₁₋₆ alkyl-N-hydroxyamino (e.g., N-methyl-N-hydroxyamino,N-ethyl-N-hydroxyamino, etc.) and so forth. More preferred is N-C₁₋₃alkyl-N-hydroxyamino, etc.

The “C₁₋₇ alkyl group” of the “C₁₋₇ alkyl group which may besubstituted” for R² includes, for example, a straight-chain or branchedC₁₋₇ alkyl group such as methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl,heptyl, etc. Among others, preferred is C₁₋₃ alkyl (e.g., methyl, ethyl,propyl, isopropyl, etc. More preferred is isopropyl.

The “substituents” of the “C₁₋₇ alkyl group which may be substituted”for R² and their number are the same as those mentioned above for the“substituents” of the “C₁₋₇ alkyl group which may be substituted” forR¹. When the number of substituents is two or more, those substituentsmay be the same as or different from one another.

The “substituents” of the “phenyl group which may be substituted” for R²includes, for example, halogen (e.g., fluoro, chloro, bromo, iodo,etc.),C₁₋₃ alkyl (e.g., methyl, ethyl, propyl, isopropyl, etc.), C₁₋₃alkoxy (e.g., methoxy, ethoxy, propoxy, isopropoxy, etc.), etc. Amongothers, preferred is halogen, more preferred is fluoro.

The “phenyl group” may have 1 to 5, preferably 1 to 3 substituents asmentioned above at possible positions and, when the number ofsubstituents is two or more, those substituents may be the same as ordifferent from one another.

R¹ is preferably a substituted branched C₃₋₇ alkyl group or asubstituted C₃₋₇ cycloalkyl group, more preferably a C₁₋₇ alkyl groupsubstituted by hydroxy or a C₃₋₇ cycloalkyl group substituted byhydroxy. Among others, preferred is a substituted C₃₋₇ cycloalkyl group.Also, a C₁₋₃ alkyl group which may be substituted by hydroxy, a C₃₋₇cycloalkyl group which may be substituted by a hydroxy, mono-C₁₋₃alkoxyamino, N-C₁₋₃ alkyl-N-hydroxyamino, hydroxyamino, is preferred.Especially preferably R¹ is (i) cyclopropyl which may be substituted bya hydroxy or (ii) methoxyamino, etc. Most preferred is cyclopropyl whichmay be substituted by a hydroxy group.

R² is preferably a C₁₋₇ alkyl group which may be substituted. Morepreferred is a C₁₋₃ alkyl group which may be substituted by a hydroxygroup. Especially preferred is isopropyl. Phenyl is also preferred.

Preferable examples of compound (I) include a compound wherein, R¹ is aC₁₋₃ alkyl group which may be substituted by a hydroxy group, a C₃₋₇cycloalkyl group which may be substituted by a hydroxy group or amono-C₁₋₃ alkoxyamino group; and

R² is a C₁₋₃ alkyl group, or a salt thereof.

More preferred is a compound wherein R¹ is (1) a C₁₋₄ alkyl groupsubstituted by 1 or 2 hydroxy, (2 ) a C₃₋₇ cycloalkyl group substitutedby hydroxy, or (3) a C₁₋₃ alkoxyamino group; and

R² is an isopropyl group or a phenyl group, or a salt thereof.

A compound wherein R¹ is (1) a C₁₋₇ alkyl group which may be substitutedby 1 or 2 substituents selected from the group consisting of hydroxy,C₁₋₃ alkyl-carbonyloxy, amino, benzyloxycarbonylamino, C₁₋₃ alkoxy, C₁₋₃alkoxy-C₁₋₃ alkoxy and C₁₋₃ alkoxy-carbonyl, (2) a C₃₋₇ cycloalkyl groupwhich may be substituted by a hydroxy group or a C₁₋₃ alkyl-carbonyloxy,or (3 ) a C₁₋₃ alkoxyamino group; and

R² is (1) an isopropyl group which may be substituted by a hydroxy groupor (2) a phenyl group, or salt thereof is also preferred.

As compound (I), concretely mentioned are

3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-cyclopropanecarbonylaminophenyl)-4-oxothieno[2,3-b]pyridineor a salt thereof,

5-benzoyl-3-(N-benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-4,7-dihydro-4-oxo-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]thieno[2,3-b]pyridine,

5-(4-fluorobenzoyl)-3-(N-benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-4,7-dihydro-4-oxo-2-(4-cyclopropanecarbonylaminophenyl)thieno[2,3-b]pyridine,

3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]-4-oxothieno[2,3-b]pyridine,

3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-N′-methoxyureidophenyl)-4-oxothieno[2,3-b]pyridine,

3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridine,

(R)-4,7-dihydro-2-[4-(3-hydroxy-2-methylpropionylaminophenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine,

4,7-dihydro-2-[4-(2-hydroxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine,

4,7-dihydro-2-[4-(3-hydroxy-3-methylbutyrylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine,

(R)-4,7-dihydro-2-[4-(2,3-dihydroxypropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine,

3-(N-benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridine,and salts thereof.

Compound (I) can be produced in any per se known manner, for example,according to the methods disclosed in WO 95/28405 or analogous methodsthereto. Concretely mentioned are the following Production method 1 andProduction method 2.

Production Method 1

Compound (I) is produced by reacting compound (II) with a compound ofthe formula: R^(1a)COOH wherein R^(1a) represents a C₁₋₇ alkyl groupwhich may be substituted or a C₁₋₇ cycloalkyl group which may besubstituted, or a salt thereof or a reactive derivative thereof[hereinafter sometimes referred to briefly as compound (III)].

The “C₁₋₇ alkyl group which may be substituted” and the “C₃₋₇ cycloalkylgroup which may be substituted” for R^(1a) are the same as thosementioned above for the “C₁₋₇ alkyl group which may be substituted” andthe “C₃₋₇ cycloalkyl group which may be substituted” for R₁,respectively.

The “reactive derivative” of the “compound of the formula: R^(1a)COOH,or a salt thereof or a reactive derivative thereof” includes, forexample, a compound of the formula:

R^(1a) COY

wherein Y represents halogen and R^(1a) is same as defined above.

The amount of compound (III) to be reacted is about 1 to 3 mol, relativeto one mol of compound (II).

This reaction is also carried out in the presence of a base. The “base”is exemplified by inorganic bases such as sodium carbonate, sodiumhydrogen carbonate, potassium carbonate, potassium hydrogen carbonate,sodium hydroxide, potassium hydroxide and thallium hydroxide, andorganic bases such as triethylamine and pyridine, etc.

The amount of the “base” is about 1 to 10 mol, preferably about 2 to 5mol, relative to one mol of compound (II).

In the case where a compound of the formula: R_(1a)COOH or a saltthereof is used, a condensing reagent which is generally used in peptidechemistry such as benzotroazol-1-yloxytripyrrolidinophosphoniumhexafluorophosphate (PyBop) may be employed to form an amide bond.

This reaction is advantageously carried out in a solvent inert to thereaction. Examples of the solvent include ethers (e.g., ethyl ether,dioxane, dimethoxyethane, tetrahydrofuran, etc.), aromatic hydrocarbons(e.g., benzene, toluene, etc.), amides (e.g., dimethylformamide,dimethylacetamide, etc.), halogenated hydrocarbons (e.g., chloroform,dichloromethane, etc.), and so forth.

The reaction temperature is usually about 0 to 150° C., preferably roomtemperature (about 15 to 25° C.). The reaction time is usually about 1to 12 hours.

Compound (II) can be produced in any per se known manner, for example,by the methods disclosed in WO 95/28405 or analogous methods thereto.

Compound (I) wherein R¹ is a C₁₋₆ alkoxyamino group which may besubstituted or a hydroxyamino group which may be substituted can also beproduced in according to the method of the following Production method2.

Production Method 2

Compound (I) is produced by reacting compound (II) withcarbonyldiimidazole (N,N′-carbonyldiimidazole; CDI), phosgene (monomer,dimer or trimer) or a chloroformate, followed by reacting with acompound of the formula: R^(1b) H wherein R^(1b) represents a C₁₋₆alkoxyamino group which may be substituted or a hydroxyamino group whichmay be substituted, or a salt thereof [hereinafter sometimes referred tobriefly as compound (IV)].

The “chloroformate” includes, for example, a compound of the formula:Cl—COOY′ wherein Y′ represents a C₁₋₆ alkyl group, such as chloroformateethyl, etc.

The “C₁₋₆ alkoxyamino group which may be substituted” and the“hydroxyamino group which may be substituted” for R^(1b) are the same asthose mentioned above for the “C₁₋₆ alkoxyamino group which may besubstituted” and the “hydroxyamino group which may be substituted” forR¹, respectively.

In the reaction of compound (II) with carbonyldiimidazole, phosgene orchloroformates, carbonyldiimidazole, phosgene or chloroformates is usedin amount of about 1 to 3 mol, relative to one mol of compound (II).

This reaction is advantageously carried out in a solvent inert to thereaction. Examples of the solvent include ethers (e.g., ethyl ether,dioxane, dimethoxyethane, tetrahydrofuran, etc.), aromatic hydrocarbons(e.g., benzene, toluene, etc.), amides (e.g., dimethylformamide,dimethylacetamide, etc.), halogenated hydrocarbons (e.g., chloroform,dichloromethane, etc.), and so forth.

The reaction temperature is usually about 0 to 150° C., preferably roomtemperature (about 15 to 25 ° C.). The reaction time is usually about 1to 36 hours.

This reaction is also carried out in the presence of a base. The “base”is exemplified by inorganic bases such as sodium carbonate, sodiumhydrogen carbonate, potassium carbonate, potassium hydrogen carbonate,sodium hydroxide, potassium hydroxide and thallium hydroxide, andorganic bases such as triethylamine and pyridine, etc.

The amount of the “base” is about 2 to 20 mol, preferably about 5 to 12mol, relative to one mol of compound (II).

The following reaction with compound (IV) can be carried out in the samecondition of the above reaction of compound (II) withcarbonyldiimidazole, phosgene or a chloroformate. The amount of compound(IV) is about 2 to 20 mol, preferably about 5 to 10 mol, relative to onemol of compound (II).

The reaction temperature is usually about 0 to 150° C., preferably roomtemperature (about 15 to 25° C.). The reaction time is usually about 1to 6 hours.

Compound (I) of the present invention may be isolated and purified byordinary means of separation such as recrystallization, distillation andchromatography, etc. When the compound of the formula (I) is obtained infree form, it can be converted to a salt by per se known methods oranalogous thereto. When compound (I) is obtained in salt form, it can beconverted to the free form or another salt by per se known methods oranalogous thereto.

Salts of compound (I) are preferably physiologically acceptable acidaddition salts. Such salts include, for example, salts with inorganicacids (e.g., hydrochloric acid, hydrobromic acid, nitric acid, sulfuricacid, phosphoric acid) and physiologically acceptable acid additionsalts with organic acids (e.g., formic acid, acetic acid,trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleicacid, citric acid, succinic acid, malic acid, methanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid). When compound (I) has anacidic group, it may form a physiologically acceptable salt with aninorganic base (e.g., alkali metals such as sodium, potassium, calciumand magnesium, alkaline earth metals, ammonia) or an organic base (e.g.,trimethylamine, triethylamine, pyridine, picoline, ethanolamine,diethanolamine, triethanolamine, dicyclohexylamine,N,N′-dibenzylethylenediamine).

Compound (I) may be a hydrate or a non-hydrate. The hydrate isexemplified by monohydrate, sesquihydrate and dehydrate.

When compound (I) is obtained as a mixture (racemate) of opticallyactive configurations, it can be resolved into the (R)- and (S)-forms bythe conventional optical resolution techniques.

Compound (I) of the present invention (hereinafter also referred to as“compound of the present invention”) possesses excellentGnRH-antagonizing activity and low toxicity. In addition, it isexcellent in oral absorbability, action sustainability, stability andpharmacokinetics. Furthermore, it can be easily produced. The compoundof the present invention can therefore be safely used in a mammal (e.g.,human, monkey, bovine, horse, dog, cat, rabbit, rat, mouse) for thepreventing and/or treating diseases depending on male or femalehormones, diseases due to excess of these hormone, etc., by suppressinggonadotropin secretion by its GnRH receptor-antagonizing action tocontrol blood sex hormone concentrations.

For example, the compound of the present invention is useful forpreventing and/or treating sex hormone-dependent cancers (e.g.,prostatic cancer, uterine cancer, breast cancer, pituitary tumor, etc.),prostatic hypertrophy, hysteromyoma, endometriosis, precocious puberty,amenorrhea, premenstrual syndrome, multilocular ovary syndrome, pimplesetc. The compound of the present invention is also useful for theregulation of reproduction in males and females (e.g., pregnancyregulators, menstruation cycle regulators, etc.). The compound of thepresent invention also be used as a male or female contraceptive, or asa female ovulation inducer. Based on its rebound effect afterwithdrawal, the compound of the present invention can be used to treatinfertility.

In addition, the compound of the present invention is useful forregulation of animal estrous, improvement of meat quality and promotionof animal growth in the field of animal husbandry. The compound of thepresent invention is also useful as a fish spawning promoter.

Although the compound of the present invention can be used alone, it iseffective to use in combination with a steroidal or non-steroidalanti-androgen agent or anti-estrogen agent. The compound of the presentinvention can also be used to suppress the transient rise in bloodtestosterone concentration (flare phenomenon) observed in administrationof a super-agonist such as Leuprorelin acetate. The compound of thepresent invention can be used in combination with a super-agonist suchas leuprorelin acetate, gonadrelin, buserelin, triptorelin, goserelin,nafarelin, histrelin, deslorelin, meterelin, lecirelin, and so forth.Among others, preferred is leuprorelin acetate. The compound of thepresent invention also may be used with a chemotherapeutic agent forcancer. A preferred example of such combination is the compound of thepresent invention in combination with chemotherapeutic agents such asifosfamide, UTF, adriamycin, peplomycin and cisplatin for prostaticcancer. For breast cancer, the compound of the present invention can beused with chemotherapeutic agents such as cyclophosphamide, 5-FU, UFT,methotrexate, adriamycin, mitomycin C and mitoxantrone.

When the compound of the present invention is used for preventing and/ortreating (as a prophylactic and/or therapeutic agent for) theabove-mentioned diseases or used in the filed of animal husbandry orfishery, it can be administered orally or non-orally, as formulated witha pharmaceutically acceptable carrier, normally in the form of solidpreparations such as tablets, capsules, granules and powders for oraladministration, or in the form of intravenous, subcutaneous,intramuscular or other injections, suppositories or sublingual tabletsfor non-oral administration. It may also be sublingually,subcutaneously, intramuscularly or otherwise administered in the form ofsustained-release preparations of sublingual tablets, microcapsules etc.Depending on symptom severity; subject age, sex, weight and sensitivity;duration and intervals of administration; property, dispensing and kindof pharmaceutical preparation; kind of active ingredient etc., dailydose is not subject to limitation. For use in the treatment of theabove-described sex hormone-dependent cancers (e.g., prostatic cancer,uterine cancer, breast cancer, pituitary tumor), prostatic hypertrophy,hysteromyoma, endometriosis, precocious puberty etc., daily dose isnormally about 0.01 to 30 mg, preferably about 0.02 to 10 mg, and morepreferably 0.1 to 10 mg, per kg weight of mammal, normally in 1 to 4divided dosages.

The above doses are applicable to the use of the compound of the presentinvention in the filed of animal husbandry or fishery. Daily dose isabout 0.01 to 30 mg, preferably about 0.1 to 10 mg, per kg weight ofsubject organism, normally in 1 to 3 divided dosages.

In the pharmaceutical composition of the present invention, the amountof compound (I) is 0.01 to 100 % by weight or so of the total weight ofthe composition.

Pharmaceutically acceptable carriers are various organic or inorganiccarrier substances in common use as pharmaceutical materials, includingexcipients, lubricants, binders and disintegrants for solidpreparations, and solvents, dissolution aids, suspending agents,isotonizing agents, buffers and soothing agents for liquid preparations.Other pharmaceutical additives such as preservatives, antioxidants,coloring agents and sweetening agents may be used as necessary.

Preferable excipients include, for example, lactose, sucrose,D-mannitol, starch, crystalline cellulose and light silicic anhydride.Preferable lubricants include, for example, magnesium stearate, calciumstearate, talc and colloidal silica. Preferable binders include, forexample, crystalline cellulose, sucrose, D-mannitol, dextrin,hydroxypropyl cellulose, hydroxypropylmethyl cellulose andpolyvinylpyrrolidone. Preferable disintegrants include, for example,starch, carboxymethyl cellulose, carboxymethyl cellulose calcium,crosslinked carmellose sodium and carboxymethyl starch sodium.Preferable solvents include, for example, water for injection, alcohol,propylene glycol, macrogol, sesame oil and corn oil. Preferabledissolution aids include, for example, polyethylene glycol, propyleneglycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane,cholesterol, triethanolamine, sodium carbonate and sodium citrate.Preferable suspending agents include, for example, surfactants such asstearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionicacid, lecithin, benzalkonium chloride, benzethonium chloride andmonostearic glycerol; and hydrophilic polymers such as polyvinylalcohol, polyvinylpyrrolidone, carboxymethyl cellulose sodium, methylcellulose, hydroxymethyl cellulose, hydroxyethyl cellulose andhydroxypropyl cellulose. Preferable isotonizing agents include, forexample, sodium chloride, glycerol and D-mannitol. Preferable buffersinclude, for example, buffer solutions of phosphates, acetates,carbonates, citrates etc. Preferable soothing agents include, forexample, benzyl alcohol. Preferable preservatives include, for example,paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethylalcohol, dehydroacetic acid and sorbic acid. Preferable antioxidantsinclude, for example, sulfites and ascorbic acid.

By adding suspending agents, dissolution aids, stabilizers, isotonizingagents, preservatives, and so forth, the compound of the presentinvention can be prepared as an intravenous, subcutaneous orintramuscular injection by a commonly known method. In such cases, thecompound of the present invention can be freeze-dried as necessary by acommonly known method. In administration to humans, for example, thecompound of the present invention can be safely administered orally ornon-orally as such or as a pharmaceutical composition prepared by mixingit with a pharmacologically acceptable carrier, excipient and diluentselected as appropriate.

Such pharmaceutical compositions include oral preparations (e.g.,powders, granules, capsules, tablets), injections, drip infusions,external preparations (e.g., nasal preparations, transdermalpreparations) and suppositories (e.g., rectal suppositories, vaginalsuppositories).

These preparations can be produced by commonly known methods in commonuse for pharmaceutical making processes.

An injection can be produced by, for example, preparing the compound ofthe present invention as an aqueous injection along with a dispersingagent (e.g., Tween 80, produced by Atlas Powder Company, USA, HCO 60,produced by Nikko Chemicals Co., Ltd., polyethylene glycol,carboxymethyl cellulose, sodium alginate), a preservative (e.g., methylparaben, propyl paraben, benzyl alcohol), an isotonizing agent (e.g.,sodium chloride, mannitol, sorbitol, glucose) and other additives, or asan oily injection in solution, suspension or emulsion in a vegetable oilsuch as olive oil, sesame oil, cottonseed oil or corn oil, propyleneglycol or the like.

An oral preparation can be produced by shaping the compound of thepresent invention by a commonly known method after addition of anexcipient (e.g., lactose, sucrose, starch), a disintegrant (e.g.,starch, calcium carbonate), a binder (e. g., starch, gum arabic,carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose),a lubricant (e.g., talc, magnesium stearate, polyethylene glycol 6000 )and other additives, and, where necessary, coating the shaped productfor the purpose of taste masking, enteric dissolution or sustainedrelease by a commonly known method. Coating agents for this purposeinclude, for example, hydroxypropylmethyl cellulose, ethyl cellulose,hydroxymethyl cellulose, hydroxypropyl cellulose, polyoxyethyleneglycol, Tween 80, Prulonic F68, cellulose acetate phthalate,hydroxypropylmethyl cellulose phthalate, hydroxymethyl cellulose acetatesuccinate, Eudragit (produced by Rohm Company, Germany; methacrylicacid/acrylic acid copolymer) and dyes (e.g., iron oxide, titaniumdioxide). For an enteric preparation, an intermediate phase may beprovided between the enteric phase and the drug-containing phase for thepurpose of separation of the two phases by a commonly known method.

An external preparation can be produced by compounding the compound ofthe present invention as a solid, semi-solid or liquid composition by acommonly known method. Such a solid composition is produced by, forexample, powdering the compound of the present invention as such or inmixture with an excipient (e.g., glycol, mannitol, starch,microcrystalline cellulose), a thickening agent (e.g., natural rubber,cellulose derivative, acrylic acid polymer) and other additives. Such aliquid composition is produced by preparing the compound of the presentinvention as an oily or aqueous suspension in almost the same manner aswith the injection. The semi-solid composition is preferably an aqueousor oily gel, or an ointment. All these compositions may contain pHregulators (e.g., carbonic acid, phosphoric acid, citric acid,hydrochloric acid, sodium hydroxide), preservatives (e.g.,paraoxybenzoic acid esters, chlorobutanol, benzalkonium chloride) andother additives.

A suppository is produced by preparing the compound of the presentinvention as an oily or aqueous solid, semi-solid or liquid compositionby a commonly known method. Useful oily bases for such compositionsinclude glycerides of higher fatty acids (e. g., cacao fat, uitepsols,produced by Dynamite Nobel Company, Germany), moderate fatty acids (e.g., MIGLYOL, produced by Dynamite Nobel Company, Germany), and vegetableoils (e.g., sesame oil, soybean oil, cottonseed oil). Aqueous basesinclude, for example, polyethylene glycols and propylene glycol. Basesfor aqueous gels include, for example, natural rubbers, cellulosederivatives, vinyl polymers and acrylic acid polymers.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is hereinafter described in more detail by meansof, but is not limited to, the following reference examples, examples,preparation examples and experimental examples.

¹H-NMR spectra are determined with tetramethylsilane as the internalstandard, using the GEMINI 200 (200 MHz) spectrometer (produced byVarian, Ltd.) the LAMBDA 300 (300 MHz) spectrometer (produced by JEOL,Ltd.) or the Bruker AM500 (500 MHz) spectrometer (produced by Bruker);all δ values are shown in ppm. Unless otherwise specifically indicated,“%” is by weight. Yield indicates mol/mol %.

The symbols used herein have the following definitions:

s: singlet

d: doublet

t: triplet

dt: double triplet

m: multiplet

br: broad

The term “at room temperature” indicates the range from about 15 to 25°C., but is not to be construed as strictly limitative.

EXAMPLES Reference Example 1 Production of2-amino-5-phenylthiophen-3-carboxylic acid ethyl ester

To a mixture of ethyl cyanoacetate (6.1 g, 50 mmol), sulfur (1.61 g, 50mmol), triethylamine (3.5 ml, 25 mmol), and dimethylformamide (10 ml),phenylacetaldehyde (50% solution in diethyl phthalate; 12.05 g, 50 mmol)was added dropwise with stirring at 45° C. over 20 minutes. Afterstirring at 45° C. for 9 hours, the reaction mixture was concentratedand the obtained residue was extracted with ethyl acetate, washed withsaline and dried (MgSO₄), the solvent was distilled off under reducedpressure. The residue was chromatographed on silica gel andrecrystallized from ether-hexane to yield pale yellow tabular crystals(5.55 g, 45%)

mp 124.5-125.5° C. (lit.; 123-124° C.).

Elemental analysis for C₁₃H₁₃NO₂S C (%) H (%) N(%)

Calculated: 63.13; 5.30; 5.66.

Found: 62.99; 5.05; 5.63.

¹H-NMR (300 MHz, CDCl₃) δ: 1.37 (3H, t, J=7.1 Hz), 4.30 (2H, d, J=7.1Hz), 5.97 (2H, br), 7.17-7.46 (6H, m).

IR (KBr): 3448, 3320, 1667, 1590, 1549 cm⁻¹.

Reference Example 2 Production of2-amino-4-methyl-5-(4-methoxyphenyl)thiophen-3-carboxylic acid ethylester

A mixture of 4-methoxyphenylacetone (16.5 g, 0.10 mol), ethylcyanoacetate (12.2 g, 0.10 mol), ammonium acetate (1.55 g, 20 mmol),acetic acid (4.6 ml, 80 mmol), and benzene (20 ml) was heated andrefluxed for 24 hours, while the water being produced was removed usinga Dean-Stark apparatus. After cooling, the reaction mixture wasconcentrated under reduced pressure and the residue was partitionedbetween dichloromethane and aqueous sodium bicarbonate. After theorganic layer was washed with saline and dried (MgSO₄), the solvent wasdistilled off under reduced pressure. To a solution in ethanol (30 ml)of the obtained residue, were added sulfur (3.21 g, 0.10 mol) anddiethylamine (10.4 ml, 0.10 mol), followed by stirring at 50 to 60° C.for 2 hours, successively the reaction mixture was concentrated. Theobtained residue was extracted with ethyl acetate, washed with salineand dried (MgSO₄), the solvent was distilled off under reduced pressure.The residue was chromatographed on silica gel and recrystallized fromether-hexane to yield light yellow tabular crystals (11.5 g, 40%).

mp 79-80° C.

Elemental analysis for C₁₅H₁₇NO₃S C (%) H (%) N (%) S (%)

Calculated: 61.83; 5.88; 4.81; 11.01.

Found: 61.81; 5.75; 4.74; 10.82.

¹H-NMR (200 MHz, CDCl₃) δ: 1.37 (3H, t, J=7.1 Hz), 2.28 (3H, s), 3.83(3H, s), 4.31 (2H, q, J=7.1 Hz), 6.05 (2H, brs), 6.91 (2H, d, J=8.8 Hz),7.27 (2H, d, J=8.8 Hz).

IR (KBr): 3426, 3328, 1651, 1586, 1550, 1505, 1485 cm⁻¹.

FAB-MS m/z: 291 (M⁺).

Reference Example 3

Using some acetone derivatives in place of 4-methoxyphenylacetone, thefollowing compound was obtained in the same manner as in ReferenceExample 2.

Yield: 40%

mp 64-65° C.

Reference Example 4 Production of[3-ethoxycarbonyl-5-(4-methoxyphenyl)-4-methylthiophen-2-yl]aminomethylenemalonicacid diethyl ester

To the compound obtained in Reference Example 2 (10 g, 34.3 mmol),ethoxymethylenemalonic acid diethyl ester (7.45 g, 34.5 mmol) was added,followed by stirring at 120° C. for 2 hours. After the reaction mixturewas cooled, the crystal precipitated was collected by filtration via theaddition of ether, again washed with ether, and dried over phosphoruspentaoxide under reduced pressure to yield yellow crystals (14.2 g,90%).

mp 122-123° C.

¹H-NMR (200 MHz, CDCl₃) δ: 1.32 (3H, t, J=7.1 Hz), 1.38 (3H, t, J=7.2Hz), 1.41 (3H, t, J=7.2 Hz), 2.34 (3H, s), 3.85 (3H, s), 4.25 (2H, q,J=7.1 Hz), 4.38 (2H, q, J=7.2 Hz), 4.45 (2H, q, J=7.2 Hz), 6.95 (2H, d,J=8.8 Hz), 7.31 (2H, d, J=8.8 Hz), 8.22 (1H, d, J=13.4 Hz), 12.74 (1H,d, J=13.1 Hz).

IR (KBr): 2984, 1720, 1707, 1688, 1653, 1599, 1518, 1499 cm⁻¹.

Reference Example 5

Using the compound obtained in Reference Example 3 as a startingmaterial, the following compound was obtained in the same manner as inReference Example 4.

Yield: 92%

mp 108-109° C.

Reference Example 6 Production of3-carboxy-5-[(4-methoxyphenyl)-4-methylthiophen-2-yl]aminomethylenemalonicacid diethyl ester

To a solution in dioxane (20 ml) of the compound obtained in ReferenceExample 4 (7.0 g, 15.2 mmol) was added, a solution of potassiumhydroxide (5.0 g, 75.7 mmol) in ethanol (30 ml) with stirring at 60 to70° C. After stirring at this temperature for 1 hour, the reactionmixture was kept standing at room temperature for 1 hour. To the mixturewas added 2 N hydrochloric acid (40 ml, 80 mmol) with ice cooling, thereaction mixture was concentrated under reduced pressure. The yellowprecipitate was collected by filtration, washed with cold water-ethanol,and dried over phosphorus pentaoxide under reduced pressure to yieldyellow powders (6.1 g, 93%).

mp 184-187° C.

¹H-NMR (200 MHz, DMSO-d₆) δ: 1.24 (3H, t, J=7.1 Hz), 1.28 (3H, t, J=7.2Hz), 2.30 (3H, s), 3.80 (3H, s), 4.15 (2H, q, J=7.1 Hz), 4.24 (2H, q,J=7.2 Hz), 7.03 (2H, d, J=8.7 Hz), 7.37 (2H, d, J=8.7 Hz), 8.08 (1H, d,J=13.6 Hz), 12.41 (1H, d, J=13.6 Hz).

IR (KBr): 3422, 2980, 1719, 1653, 1607, 1551, 1512 cm⁻¹.

Reference Example 7

Using the compound obtained in Reference Example 5 as a startingmaterial, the following compound was obtained in the same manner as inReference Example 6.

Yield: 98 %

mp 187-190 OC.

Reference Example 8 Production of4-hydroxy-2-(4-methoxyphenyl)-3-methylthieno[2,3-b]pyridine-5-carboxylicacid ethyl ester

With stirring at 120° C., small portions of the compound obtained inReference Example 6 (6.0 g, 13.8 mmol) was added to polyphoshoric acidester (PPE) (90 ml). After stirring at the same temperature for 30minutes, the reaction mixture was poured into ice water and extractedwith ethyl acetate. The combined extracts were washed with saline, anddried (MgSO₄), the solvent was distilled off under reduced pressure. Theobtained residue was chromatographed on silica gel to yield yellowpowders (3.65 g, 77%). For a sample for elemental analysis, the powdersobtained were recrystallized from ethanol to yield yellow crystals.

mp 162-163° C.

Elemental analysis for C₁₈H₁₇NO₄S C (%) H (%) N (%) S (%)

Calculated: 62.96; 4.99; 4.08; 9.34.

Found: 62.89; 5.04; 4.01; 9.34.

¹H-NMR (200 MHz, CDCl₃) δ: 1.47 (3H, t, J=7.1 Hz), 2.63 (3H, s), 4.87(3H, s), 4.49 (2H, q, J=7.1 Hz),6.99 (2H, d, J=8.8 Hz), 7.44 (2H, d,J=8.8 Hz), 8.84 (1H,s), 12.11 (1H, s).

IR (KBr): 3434, 2992, 1692, 1601, 1582, 1535, 1504 cm⁻¹.

FAB-MS m/z: 344 (MH⁺).

Reference Example 9

Using the compound obtained in Reference Example 7 as a startingmaterial, the following compound was obtained in the same manner as inReference Example 8.

Yield: 60%

mp 155-157° C.

Reference Example 10 Production of4-hydroxy-2-(4-nitrophenyl)-3-methylthieno[2,3-b]pyridine-5-carboxylicacid ethyl ester

To a solution in concentrated sulfuric acid (10 ml) of the compoundobtained in Reference Example 9 (3.76 g, 12.0 mmol) was added dropwise asolution of sodium nitrate (1.27 g, 15.0 mmol) in concentrated sulfuricacid (5 ml) with ice cooling. After stirring at this temperature for 30minutes, the reaction mixture was poured into ice water and extractedwith chloroform. The combined extracts were washed with saline, anddried (MGSO₄), the solvent was distilled off under reduced pressure. Theobtained residue was chromatographed on silica gel to yield yellowpowders, which was recrystallized from ethanol to yield yellow crystals(1.75 g, 41%).

mp 260-261° C.

Elemental analysis for C₁₇H₁₄N₂O₅S C (%) H (%) N (%)

Calculated: 56.98; 3.94; 7.82.

Found: 56.66; 3.91; 7.86.

¹H-NMR (200 MHz, CDCl₃) δ: 1.49(3H, t, J=7.1 Hz), 2.70(3H, s), 4.51 (2H,q, J=7.1 Hz), 7.70(2H, d, J=8.8 Hz), 8.34(2H, d, J=8.8 Hz), 8.89(1H,s),12.27(1H, s).

IR (KBr): 3002, 1692, 1605, 1514, 1350, 1290 cm⁻¹.

FAB-MS m/z: 358 (MH⁺).

Reference Example 11 Production of4,7-dihydro-7-(2-methoxybenzyl)-2-(4-methoxyphenyl)-3-methyl-4-oxothieno[2,3-b]pyridine-5-carboxylicacid ethyl ester

To a suspension of sodium hydride (60% dispersion in oil; 123 mg, 3.08mmol) in dimethylformamide (3 ml) was added a solution of the compoundobtained in Reference Example 8 (1.0 g, 2.91 mol) in dimethylformamide(20 ml) under nitrogen stream with ice cooling. After stirring at thistemperature for 30 minutes, to the mixture was added dropwise a solutionof 2-methoxybenzyl chloride (0.92 g, 5.87 mmol) in dimethylformamide (3ml). After stirring at room temperature for 23 hours and at 70° C. for 2hours, the reaction mixture was concentrated, and then the obtainedresidue was partitioned between ethyl acetate and an aqueous solution ofammonium chloride. The water layer was extracted with ethyl acetate. Thecombined extracts were washed with saline, and dried (MgSO₄), thesolvent was distilled off under reduced pressure. The obtained residuewas chromatographed on silica gel to yield light yellow amorphouspowders (0.95 g, 70%). For a sample for elemental analysis, the obtainedpowders were recrystallized from dichloromethane-ether to yield yellowprismatic crystals.

mp 165-167° C.

Elemental analysis for C₂₆H₂₅NO₅S 0.5H₂O C (%) H (%) N (%)

Calculated: 66.08; 5.55; 2.96.

Found: 66.33; 5.44; 2.74.

¹H-NMR (200 MHz, CDCl₃) δ: 1.41(3H, t, J=7.1 Hz), 2.65(3H, s), 3.85(3H,s), 3.86(3H, s), 4.39(2H, q, J=7.1 Hz), 5.16(2H, s), 6.92-7.00(4H, m),7.21-7.41 (4H, m), 8.41 (1H, s).

IR (KBr): 2980, 1727, 1684, 1609, 1590, 1497, 1464 cm⁻¹.

Reference Example 12

The following compound was produced by the same method as described inReference Example 11, using benzyl 2,6-difluorochloride in place of2-methoxybenzyl chloride with the compound obtained in Reference Example10 as the starting material.

Yield: 81%

mp 215-217° C.

Reference Example 13 Production of3-bromomethyl-4,7-dihydro-7-(2-methoxybenzyl)-2-(4-methoxyphenyl)-4-oxothieno[2,3-b]pyridine-5-carboxylicacid ethyl ester

A mixture of the compound obtained in Reference Example 11 (0.35 g,0.755 mmol), N-bromosuccinimide (0.135 g, 0.758 mmol,)α,α,′-azobisisobutyronitrile (13 mg, 0,079 mmpl), and carbontetrachloride (5 ml) was heated and refluxed for 2 hours. After cooling,the insoluble substances were filtered off, and then the filtrate wasdiluted with chloroform. After the organic layer was washed with salineand dried (MgSO₄), the solvent was distilled off under reduced pressure.The obtained residue was recrystallized from ethyl acetate to yieldcolorless needles (0.272 g, 66%).

mp 200-201° C.

Elemental analysis for C₂₆H₂₄NO₅SBr C (%) H (%) N (%)

Calculated: 57.57; 4.46; 2.58.

Found: 57.75; 4.31; 2.31.

¹H-NMR (200 MHz, CDCl₃) δ: 1.40(3H, t, J=7.1 Hz), 3.86(6H, s), 4.40(2H,q, J=7.1 Hz), 5.05(2H, s), 5.16(2H, s), 6.92-7.04(4H, m), 7.23-7.28(1H,m), 7.34-7.43(1H, m), 7.57(2H, d, J=8.9 Hz), 8.46(1H, s).

IR (KBr): 2980, 1725, 1607, 1588, 1497 cm⁻¹.

Reference Example 14

Using the compounds obtained in Reference Examples 12, 22 and 27 as astarting material, respectively, the following compounds (ReferenceExamples 14-1 to 14-3) were obtained in the same manner as in ReferenceExample 13.

Reference Example 14-1

Yield: 81%

mp 200-202° C.

Reference Example 14-2

Yield: 80%

Amorphous.

Reference Example 14-3

Yield: 79%

mp 189-192° C.

Reference Example 15 Production of3-benzylaminomethyl-4,7-dihydro-7-(2-methoxybenzyl)-2-(4-methoxyphenyl)-4-oxothieno[2,3-b]pyridine-5-carboxylicacid ethyl ester hydrochloride

To a solution of the compound obtained in Reference Example 13 (0.245 g,0.452 mmol) in dimethylformamide (5 ml) were added triethylamine (0.10ml, 0.717 mmol) and benzylamine (80 μl, 0.732 mmol) with ice cooling.After stirring at room temperature for 1.5 hours, the reaction mixturewas concentrated, and then the obtained residue was partitioned betweenethyl acetate and saturated aqueous sodium bicarbonate. The water layerwas extracted with ethyl acetate. The combined organic extracts weredried (MgSO₄), the solvent was distilled off under reduced pressure. Theobtained residue was chromatographed on silica gel to yield a colorlessoil (0.135 g, 53%). To a solution of this oil in ethanol (4 ml) wasadded 10 N ethanolic hydrochloric acid (0.2 ml) with ice cooling,followed by stirring at this temperature for 10 minutes. The reactionmixture was concentrated, and then the obtained residue was crystallizedfrom ethyl acetate-ether to yield the hydrochloride (0.113 g) as whitecrystals.

mp [Hydrochloride] 118-119° C.

Elemental analysis for C₃₃H₃₂N₂O₅S HCl 0.9H₂O C (%) H (%) N (%)

Calculated: 63.79; 5.64; 4.51.

Found: 64.03; 5.44; 4.51.

¹H-NMR (300 MHz, CDCl₃) [Free amine] δ: 1.40(3H, t, J=7.1 Hz), 2.05(1H,br),3.81(3H, s), 3.86(3H, s), 3.87(2H, s), 3.94(2H, s), 4.40(2H, q,J=7.1 Hz), 5.18(2H, s), 6.80(2H, d, J=8.8 Hz), 6.91-6.99(2H, m),7.20-7.42(9H, m), 8.45(1H, s).

IR (KBr) [Hydrochloride]: 3422, 2938, 1719, 1605, 1560, 1545, 1502, 1460cm⁻¹.

Reference Example 16 Production of4-methyl-2-[(4-methyl-3-oxo-1-penten-1-yl)amino]-5-phenylthiophen-3-carboxylicacid ethyl ester

The compound obtained in Reference Example 3 (10 g),1-methoxy-4-methyl-1-penten-3-one of 85% content (6.9 g),p-toluenesulfonic acid monohydrate (0.219 g), and toluene (100 ml) weremixed and stirred at room temperature for 2.5 hours. After the reactionmixture was diluted with ethyl acetate and washed with asaturatedaqueous solution of sodium hydrogen carbonate, the water layer wasextracted with ethyl acetate. The combined extracts were washed withsaturated saline, the organic layers were dried over anhydrous magnesiumsulfate. The residue was crystallized via the addition of a seedcrystal, after which it was triturated with hexane, collected byfiltration, and washed, to yield the title compound (12.64 g, 92%).

mp 104-108° C.

Reference Example 17 Production of4,7-dihydro-7-(2-methoxybenzyl)-2-(4-methoxyphenyl)-3-methyl-4-oxothieno[2,3-b]pyridine-N-benzylpiperazinyl-5-carboxamide

To 1-benzylpiperazine (0.77 g, 4.37 mmol) was added dropwise a solutionof diisobutyl aluminum hydride (DIBAL) in toluene (1.5 N, 2.9 ml, 4.37mmol) with ice cooling. After the addition was completed, the solutionwas allowed to warm to room temperature and stirred for further 0.5hours. To this solution was added a solution of the compound obtained inReference Example 11 (0.50 g, 1.08 mmol) in toluene (5 ml) at roomtemperature. After the mixture was further stirred at room temperaturefor 15 hours, methylene chloride (30 ml) was added. After water washing,the solution was dried over sodium sulfate, and then the solvent wasconcentrated under reduced pressure to yield a solid (1.03 g), which wasrecrystallized from methylene chloride-n-hexane to yield the titlecompound (0.48 g, 78%).

mp 233-235° C.

Elemental analysis for C₃₅H₃₅N₃O₄S 1/2H₂O C (%) H (%) N (%) S (%)

Calculated: 69.75; 6.02; 6.97; 5.32.

Found: 69.88; 6.06; 6.98; 5.39.

¹H-NMR (300 MHz, CDCl₃) δ: 2.45-2.55(4H, m), 2.63(3H, s), 3.43-3.49(2H,m), 3.55(2H, s), 3.73-3.82(2H, m), 3.84(6H, s), 5.11(2H, s),6.89-6.98(4H, m), 7.21-7.40(9H, m), 7.79(1H,s) .

Reference Example 18

Using the compound obtained in Reference Example 12 as a startingmaterial and N,O-dimethylhydroxy in place of 1-benzylpiperazine, thefollowing compound was obtained in the same manner as in ReferenceExample 17.

Yield: 80%

mp 223-224° C.

Reference Example 19 Production of4-hydroxy-5-isobutyryl-3-methyl-2-phenylthieno[2,3-b]pyridine

The compound obtained in Reference Example 16 (50 g) and diphenyl ether(500 ml) were mixed, and then this mixture was heated and refluxed for 4hours, while the ethanol being produced with the progress of thereaction was distilled off. After cooling, the diphenyl ether wasdistilled off under reduced pressure, and then the crude crystalprecipitated was washed with n-hexane to yield the title compound (35.1g, 81).

mp 114-117° C.

Reference Example 20

Using the compound obtained in Reference Example 14-1 as startingmaterial and N-methylbenzylamine in place of benzylamine, the followingcompounds (Reference Examples 20-1 to 20-3) were obtained in the samemanner as in Reference Example 15.

Reference Example 20-1

Yield: 83%

mp 140-144° C.

Reference Example 20-2

Yield: 91%

mp 145-147° C.

Reference Example 20-3

Yield: 78%

mp 175-177° C.

Reference Example 21

Using the compounds obtained in Reference Example 20 as startingmaterials, the following compounds (Reference Examples 21-1 to 21-3)were obtained in the same manner as in Reference Example 28.

Reference Example 21-1

Yield: 79%

mp 158-160° C.

Reference Example 21-2

Yield: 96%

mp 195-196° C.

Reference Example 21-3

Yield: 71%

mp 144-146° C.

Reference Example 22 Production of7-(2,6-difluorobenzyl)-4,7-dihydro-5-isobutyryl-3-methyl-4-oxo-2-phenyl-thieno[2,3-b]pyridine

The compound obtained in Reference Example 19 (35 g), potassiumcarbonate ( 18.6 g), and N,N-dimethylformamide (280 ml) were mixed, andthen 2,6-difluorobenzyl bromide ( 27.9 g) was added, followed bystirring at room temperature for 4 hours. To the reaction mixture wasadded dropwise water (560 ml), followed by stirring for 30 minutes, andthen the mixture was stirred for 1 hour with ice cooling. The crudecrystal was collected by filtration, washed with water, and dried,successively suspended in a 1:1 mixed solvent (250 ml) of ethyl acetateand diisopropyl ether and stirred at 25 to 40° C. for 1 hour, thenstirred with ice cooling for 1 hour, the residual crystal was collectedby filtration and washed with the above mixed solvent (125 ml) to yieldthe title compound (44.6 g, 92%).

mp 205-207° C.

Reference Example 23

Using the compounds obtained in Reference Example 14-2 and ReferenceExample 24 as starting materials and N-methylbenzylamine in place ofbenzylamine, the following compounds (Reference Examples 23-1 to 23-2)were obtained in the same manner as in Reference Example 15.

Reference Example 23-1

Yield: 83%

mp 197-199° C.

Reference Example 23-2

Yield: 66%

mp 151-152° C.

Reference Example 24 Production of3-bromomethyl-7-(2,6-difluorobenzyl)-4,7-dihydro-5-isobutyryl-2-(4-nitrophenyl)-4-oxothieno[2,3b]pyridine

After the compound obtained in Reference Example 14-3 (1 g) wasdissolved in methanesulfonic acid (5 ml) with cooling at 10 to 12° C., asolution of sodium nitrate (0.165 g) in methanesulfonic acid (2.5 ml)was added dropwise to this mixture. After being stirred as was for 2hours, the mixture was poured into cold water, and then the crystalprecipitated was collected by filtration, washed with water anddiisopropyl ether, and dried to yield a crude product (1.04 g), whichwas then triturated with ethyl acetate (15 ml), the cooled with ice,filtered, and washed with cold ethyl acetate to yield the title compound(0.647 g, 59.5%).

mp 202 to 204° C. (recrystallized from methanol).

Reference Example 25 Production of4,7-dihydro-7-(2,6-difluorobenzyl)-2-(4-nitrophenyl)-3-(N-benzyl-N-methlyaminomethyl)-4-oxothieno[2,3-b]pyridinie-5-(N-isopropyl)carboxamide

To a solution of isopropylamine (0.296 g, 5 mmol) on anhydrous methylenechloride (5 ml) was added dropwise a solution of trimethylaluminum inhexane (15%, 2.41 ml, 5.0 mmol) at 0° C. After the addition wascompleted, the solution was allowed to warm to room temperature andfurther stirred for 1 hour. To this solution was added a solution of thecompound obtained in Reference Example 20-2 (0.12 g, 0.25 mmol) inanhydrous methylene chloride (3 ml) with ice cooling (0° C.) over 30minutes. After the mixture was stirred at room temperature for further 1hour, chloroform (50 ml) was added, followed by washing with water. Thecombined organic layers were dried with sodium sulfate, and then thesolvent was concentrated under reduced pressure to yield a solid, whichwas recrystallized from chloroformethyl acetate-ethyl ether to yieldcolorless crystals (0.0965 g, 70%).

mp 200-202° C.

¹H-NMR (500 MHz, CDCl ₃) [Free amine] δ: 1.30(6H, d, J=6.7 Hz) ,2.15(3H, s), 3.66(2H, s), 4.18(2H, s), 4.18-4.31(1H, m), 5.32(2H, s),7.00(2H, t, J=7.26 Hz), 7.13-7.25(5H, m), 7.42(1H, t, J=7.3 Hz),8.02(2H, d, J=8.9 Hz), 8.26(2H, d, J=8.9 Hz), 8.73(1H, s), 10.02(1H, d,J=9.1 Hz).

IR (KBr): 2974, 1661, 1597, 1547, 1497, 1346, 1212, 1035 cm⁻¹

FAB-Mass m/z 617(MH)⁺

Reference Example 26

Using the compound obtained in Reference Example 20-2 as a startingmaterial and N,O-dimethylhydroxyamine, the following compound wasobtained in the same manner as in Reference Example 25.

Yield: 96%

mp 100-102° C.

Reference Example 27 Production of4,7-dihydro-7-(2,6-difluorobenzyl)-2-(4-nitrophenyl)-5-benzoyl-3-methyl-4-oxothieno[2,3-b]pyridine

The compound obtained in Reference Example 18 (3.93 g, 7.87 mmol) wasdissolved in anhydrous tetrahydrofuran (THF) (300 ml) with slightlywarming. While this solution was kept at 0° C., a solution ofphenylmagnesium bromide in THF (1 M, 15.7 ml, 15.7 mmol) was addeddropwise to the mixture over 10 minutes. After the addition wascompleted, the solution was stirred for further 1 hour. The reactionmixture was partitioned between ethyl acetate (300 ml) and water (50ml), and then the water layer was again extracted with ethyl acetate.The combined organic layers were dried with magnesium sulfate, thesolvent was concentrated under reduced pressure to yield a residue,which was chromatographed on silica gel to yield yellow crystals (3.00g, 74%) , which was then recrystallized from ethyl acetate-hexane togive yellow crystals.

mp 114-116° C.

Elemental analysis for C₂₈H₁₈N₂O₄SF₂ 0.7H₂O C (%) H (%) N (%)

Calculated: 63.56; 3.70; 5.29.

Found: 63.83; 3.95; 5.08.

H-NMR (500 MHz, CDCl₃) δ: 2.68(3H, s), 5.30(2H, s), 7.02(2H, t, J=8.1Hz), 7.43(3H, t, J=7.2 Hz), 7.52-7.63(3H, m), 7.86(2H, d, J=7.5 Hz),7.99(1H, s), 8.30(2H, d, J=8.7 Hz).

IR (KBr): 3422, 3068, 1665, 1615, 1491, 1473, 1346, 853 cm⁻¹.

FAB-Mass m/z 517(MH)⁺

Reference Example 28 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2,6-difluorobenzyl)-2-(4-aminophenyl)-5-benzoyl-4-oxothieno[2,3-b]pyridine

To a mixture of the compound obtained in Reference Example 23-1 (0.30 g,0.47 mmol) in ethyl alcohol (6 ml) were added water (2 ml), and then onedrop of concentrated hydrochloric acid was added to yield a uniformsolution. To this solution were added iron powder (0.105 g, 2.0 mmol)and concentrated hydrochloric acid (0.39 ml, 4.7mmol) carefully. Afterthe addition was completed, the mixture was stirred at room temperaturefor 5 hours and filtered through Celite. A small amount of aqueousammonia was added, and then the reaction filtrate was concentrated underreduced pressure. The obtained residue was poured into ice water,neutralized with sodium bicarbonate, and extracted with ethyl acetate.After the combined organic layers were washed with saline, and dried(MgSO₄), the solvent was distilled off under reduced pressure. Theobtained residue was chromatographed on silica gel and recrystallizedfrom isopropyl ether to yield yellow needles (0.24 g, 84%).

mp 126-128° C.

Elemental analysis for C₃₆H₂₉N₃O₂SF₂ 1/2H₂O C (%) H (%) N (%)

Calculated: 68.93; 5.04; 6.70.

Found: 68.71; 5.18; 6.62.

¹H-NMR (300 MHz, CDCl₃) δ: 2.13 (3H, s), 3.65 (2H, s), 3.87(2H, br s),4.14(2H, s), 5.28(2H, s), 6.74(2H, d, J=8.7 Hz), 7.00(2H, t, J=7.8 Hz),7.16-7.24(5H, m), 7.36-7.46(3H, m), 7.53(1H, t, J=7.2 Hz), 7.62(2H, d,J=8.4 Hz), 7.89(2H, d, J=7.2 Hz), 7.94(1H, s).

IR (KBr): 3358, 1607, 1495, 1473, 1035 cm⁻¹.

FAB-Mass m/z 606(MH)⁺

Reference Example 29 Production of2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-4,7-dihydro-5-isobutyryl-3-(N-benzyl-N-methylaminomethyl)-4-oxothieno[2,3-b]pyridine

To a solution of the compound obtained in Reference Example 23-2 (0.25g, 0.415 mmol) in methanol (5 ml) were added dropwise iron powder (0.093g, 1.66 mmol) and concentrated hydrochloric acid (0.8 ml) with icecooling. After the addition was completed, the mixture was stirred atroom temperature for 1 hour and filtered through Celite. A small amountof a saturated aqueous solution of sodium hydrogen carbonate (10 ml) wasadded, followed by extraction with methylene chloride (30 ml×3). Thecombined extracts were washed with saline, and dried (MgSO₄), thesolvent was distilled off under reduced pressure. The obtained residuewas chromatographed on silica gel to yield light yellow amorphouspowders (0.203 g, 86%).

¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H, d), 2.11(3H, s), 3.65(2H, s),3.85(2H, br s), 4.17(2H, s), 4.18(1H, m), 5.25(2H, s), 6.73(2H, d),6.95(2H, t), 7.10-7.26(5H, m), 7.42(1H, m), 7.58(2H, d), 8.27(1H, s).

Reference Example 30 Production of5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihyrdro-3-(N-benzyl-N-methylaminomethyl)-2-(4-propionylaminophenyl)-4-oxothieno[2,3-b]pyridine

The compound obtained in Reference Example 28 (0.14 g, 0.23 mmol) wasdissolved in anhydrous methylene chloride (2 ml), and then to themixture was added triethylamine (0.038 ml) with ice cooling (0° C.).After this solution was stirred for a while, to the mixture was addedpropionyl chloride (0.021 ml, 0.243 mmol). After the addition wascompleted, the solution was further stirred with ice cooling (0° C.) for40 minutes. The reaction mixture was partitioned between methylenechloride (25 ml) and very thinly overlaid water (10 ml). The water layerwas again extracted with methylene chloride (25 ml). The combinedorganic layers were washed with saline, and dried (MgSO₄), the solventwas distilled off under reduced pressure. The solid obtained wasrecrystallized from ethyl acetate-isopropyl ether to yield yellowneedles (0.10 g, 65%).

mp 226-228° C.

Elemental analysis for C₃₉H₃₃N₃OSF₂ 0.7H₂O C (%) H (%) N (%)

Calculated: 69.46; 5.14; 6.23.

Found: 69.60; 5.18; 6.04.

Thus obtained compound was dissolved in ethyl acetate, and then asaturated solution of hydrogen chloride (HCl) in ether was added inequal to slightly excess amount, and then the crystal precipitated wasrecrystallized from isopropyl ether to yield light yellow needles (0.095g, 61%).

mp 218-220° C.

Elemental analysis for C₃₉H₃₃N₃O₃SF₂ HCl 3.5H₂O C (%) H (%) N (%)

Calculated: 61.53; 5.43; 5.52.

Found: 61.83; 5.33; 5.30.

¹H-NMR (300 MHz, DMSO-d₆) δ: 1.11(3H, t, J=7.2 Hz),1.93(3H, s) 2.35(2H,q,J=7.5 Hz), 3.44(2H, s), 4.00(2H, s), 5.62(2H, s), 7.11-7.25(6H, m),7.43-7.72(10H, m), 7.79(2H, d, J=7.5 Hz), 8.40(1H, s), 10.03(1H, s).

IR (KBr): 3422, 3068, 1603, 1502, 1473, 1035 cm⁻.

FAB-Mass m/z 662(MH)⁺

Reference Example 31

The following compounds (Reference Examples 31-1 through 31-10) wereproduced by the same method as described in Reference Example 30, usingvarious acid chlorides, isocyanates, and chlorocarbonic acid esters inplace of propionyl chloride, and also using the amines shown in Example16 and carbonyldiimidazole, with the compounds obtained in ReferenceExamples 28 and 29 as the starting materials. The following compounds(Reference Examples 31-11 through 31-16) can also be produced in thesame manner.

Reference Example 31-1-1

Yield: 68%

mp 238-240° C.

Reference Example 31-1-2

mp 230-232° C.

Reference Example 31-2-1

Yield: 64%

mp 201-204° C.

Reference Example 31-2-2

mp 207-214° C.

Reference Example 31-3-1

Yield: 55%

mp 207-210° C.

Reference Example 31-3-2

mp 222-226° C.

Reference Example 31-4

Yield: 49%

mp 185-187° C.

Reference Example 31-5

Yield: 79%

mp 216-218° C.

Reference Example 31-6

Yield: 73%

mp 180-183° C.

Reference Example 31-7

Yield: 65%

mp 245-247° C.

Reference Example 31-8

Yield: 65%

Reference Example 31-9

Yield: 70%

mp 232-234° C.

Reference Example 31-10

Yield: 73%

mp 192-197° C.

Reference Example 31-11

Reference Example 31-12

Reference Example 31-13

Reference Example 31-14

Reference Example 31-15

Reference Example 31-16

Reference Example 32 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihyrdro-7-(2,6-difluorobenzyl)-2-(4-nitrophenyl)-5-benzoyl-4-oxothieno[2,3-b]pyridine

The compound obtained in Reference Example 26 (1.91 g, 3.09 mmol) wasdissolved in anhydrous tetrahydrofuran (THF) (30 ml) with warming, andthen to the mixture was added dropwise a solution of phenylmagnesiumbromide in THF (1 M, 6.18 ml, 6.2 mmol) with ice cooling (0° C.) over 10minutes. After stirring under ice cooling conditions for 1 hour, thereaction mixture was partitioned between ethyl acetate (100 ml) andaqueous hydrochloric acid (0.5 N, 100 ml), and then the organic layerwas again washed with saturated saline (100 ml). After the organic layerwas dried (MgSO₄), the solvent was distilled off under reduced pressure.The obtained residue was chromatographed on silica gel to yield yellowcrystals (1.00 g, 51%), which was then recrystallized from isopropylether to yield yellow needles.

mp 197-199° C.

Elemental analysis for C₃₆H₂₇N₃O₄SF₂ 0.7H₂O C (%) H (%) N (%)

Calculated: 66.70; 4.42; 6.48.

Found: 66.59; 4.48; 6.42.

¹ H-NMR (300 MHz, CDCl₃) δ: 2.17(3H, s), 3.61(2H, s), 4.16(2H, s),5.30(2H, s), 7.03(2H, t, J=8.1 Hz), 7.19-7.25(5H, m), 7.40-7.47( 3H, m),7.56 (1H, t, J=7.5 Hz), 7.88(2H, d, J=6.9 Hz) 7.96(1H, s), 8.10(2H, d,J=8.7 Hz), 8.28(2H, d, J=8.7 Hz).

IR (KBr): 3430, 1663, 1611, 1518, 1473, 1348, 853 cm⁻¹.

FAB-Mass m/z 636(MH)⁺

Reference Example 33 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihyrdro-7-(2,6-difluorobenzyl)-2-(4-isobutyrylaminophenyl)-5-(2-bromoisobutyryl)-4-oxothieno[2,3-b]pyridine

The compound obtained in Reference Example 31-10 (0.48 g, 0.75 mmol) wasdissolved in dichloromethane (15 ml). To the solution was added dropwise47% hydrobromic acid (0.35 ml, 3.0 mmol) at room temperature for 1minute, followed by further stirring for 10 minutes. To this reactionmixture was added dropwise bromine (39 μl, 0.75 mmol), followed byfurther stirring at room temperature for 24 hours. The reaction mixturewas partitioned between dichloromethane (30 ml) and aqueous sodiumbicarbonate (saturated, 60 ml), and then the organic layer was againwashed with saturated saline (100 ml). After the organic layer was dried(MgSO₄), the solvent was distilled off under reduced pressure to yieldbrown amorphous crystals (0.53 g).

Reference Example 34 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihyrdro-7-(2,6-difluorobenzyl)-2-(4-isobutyrylaminophenyl)-5-methacryloyl-4-oxothieno[2,3-b]pyridine

The compound obtained in Reference Example 33 (0.52 g, 0.72 mmol) wasdissolved in dimethylformamide (DMF) (30 ml), and then to this solutionwas added potassium acetate (2.0 g, 20 mmol) at room temperature,followed by stirring at 100° C. for 20 hours. The reaction mixture waspartitioned between ethyl acetate (30 ml) and water (30 ml), and thenthe water layer was again washed with ethyl acetate (10 ml). Thecombined orgain layers were again washed with saturated saline (30 ml).After the orgain layer was dried (mgSO₄), the solvent was distilled offunder reduced pressure to yield a brown oil (0.56 g).

Example 1 Example 1-1 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-cyclopropanecarbonylaminophenyl)-4-oxothieno[2,3-b]pyridine

To a solution of the compound obtained in Reference Example 29 (2.57 g,4.5 mmol) in dichloromethane (50 ml) were added cyclopropanecarboxylicacid (861 mg, 10 mmol) and diisopropylethylamine (2.59 g, 20 mmol), andthen benzotriazol-1-yl-oxo tripyrrolidino phosphoniumhexafluorophosphoric acid (PhBop) (5.40 g, 10 mmol) was added to it in asmall portion with ice cooling. After stirring at room temperature for 1day, the reaction mixture was poured into a 0.1 N aqueous solution ofpotassium hydroxide and extracted with cholorform. The combined extractswere washed with saline, and dried (Na₂SO₄), the solvent was distilledoff under reduced pressure. The obtained residue was chromatographed onsilical gel and recrystallized from ethyl acetate to yield white powderycrystals (2.15 g, 79%).

¹H-NMR (300 MHz, CDCl₃) δ: 0.85-0.90(2H,m), 1.10-1.15(2H,m), 1.18(6H,d), 1.53-1.59(1H, m), 2.09(3H, s), 4.12-4.20(1H, m), 4.16(2H, s),5.25(2H, s), 6.99(2H, t), 7.10-7.25(6H, m), 7.35-7.45(1H, m), 7.61(2H,d), 7.67(1H, br s), 7.76(2H, d), 8.27(1H, s).

FAB-Mass m/z 608(MH)⁺

Example 1-2 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-cyclopropanecarbonylaminophenyl)-4-oxothieno[2,3-b]pyridinehydrochloride

The white powdery crystals obtained in Example 1-1 were dissolved inethanol, and then a solution of hydrogen chloride in ethyl ether wasadded to it in a small excess amount, followed by recrystallization fromethanol-ethyl ether, to yield white powdery crystals (2.20 g, 72%).

mp 254-256° C.

Elemental analysis for C₃₇H₃₅N₃O₃SF₂ HCl C (%) H (%) N (%)

Calculated: 65.72; 5.37; 6.21.

Found: 65.61; 5.38; 6.25.

IR (KBr): 2950, 1673, 1595, 1502, 1473, 1408, 1313, 1183 cm⁻¹.

Example 2 Production of4,7-dihydro-2-[4(3-acetoxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylamino)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

To a solution of the compound obtained in Reference Example 29 (1.14 g,2.0 mmol) in dichloromethane (30 ml) were added dropwise triethylamine(500 mg, 5 mmol) and 3-acetoxyisobutyrylchloride (0.5 g, 3 mmol) withice cooling over 1 minute, followed by further stirring at roomtemperature for 1 hour. The reaction mixture was paartitioned between asaturated aqueous solution of sodium hydrogen carbonate (30 ml) anddichloromethane (30 ml), and then the water layer was again extractedwith dichloromethane (10 ml). The combined extracts were washed withsaline, and dried (Na₂SO₄), the solvent was distilled off under reducedpressure. The obtained residue was chromatographed on silica gel toyield brown crystals (1.4 g).

¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H,d), 1.30(3H,d), 2.08(3H,s),2.12(3H,s), 2.73-2.84(1H,m), 3.65(2H,s), 4.11-4.36(3H,m), 4.17(2h,s),5.27(2H,s), 7.00(2H,t), 7.13-7.22(5H,m), 7.36-7.46(1H,m), 7.52(1H,br,s), 7.62-7.82(4H,q), 8.29(1H, s).

Example 3 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]-4-oxothieno[2,3-b]pyridine

The compound obtained in Example 2 (1.40 g) was dissolved in methanol(100 ml), and then to the solution was added potassium carbonate (2.0 g,15 mmol), followed by stirring at room temperature for 15 minutes. Thereaction mixture was partitioned between ethyl acetate (200 ml) anddistilled water (300 ml), and then the water layer was extracted withethyl acetate (100 ml). The combined extracts were washed with saline,and dried (Na₂SO₄), the solvent was distilled off under reducedpressure. The obtained residue was chromatographed on silica gel andrecrystallized from ether to yield white crystals (0.95 g, 72%).

mp 161-162° C.

Elemental analysis for C₃₇H₃₇N₃O₄SF₂ 0.5H₂O C (%) H (%) N (%)

Calculated: 66.65; 5.74; 6.30.

Found: 66.70; 5.52; 6.40.

¹H-NMR (300 MHz, CDCl₃) δ: 1.17(6H,d), 1.18(3H,d), 2.06(3H,s),2.67-2.78(1H,m), 3.61(2H,s), 3.57-3.84(2H,m), 3.99-4.08(1H,m),4.15(2H,s), 5.52(2H,s), 7.08(2H,t), 7.15(5H,s), 7.44-7.54(1H,m),7.58(2H,d), 7.69(2H,d), 8.46(1H, s).

FAB-Mass m/z 658(MH)⁺

Example 4

Using the compound obtained in Reference Example 29, the followingcompounds (Examples 4-1 to 4-3) were obtained in the same manner as inExample 1.

Example 4-1

Yield: 67%

mp 201-203° C.

Example 4-2

Yield 58%

mp 135-137° C.

Example 4-3

Yield: 75%

mp 205-207° C.

Example 5 Example 5-1 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2,6-difluorobenzyl)-2-[4-(2-hydroxyacetylamino)phenyl]-5-isobutyryl-4-oxothieno[2,3-b]pyridine

The compound obtained in Example 4-1 (0.137 g) was dissolved indichloromethane (1.0 ml), and then to the solution was added dropwisetrifluoroacetic acid (1.0 ml) with ice cooling. After stirring under icecooling conditions for 1 hour, the reaction mixture was concentrated todryness under reduced pressure, and then the obtained residue waspartitioned between chloroform (50 ml) and saturated aqueous sodiumbicarbonate (50 ml), and then the water layer was again extracted withchloroform (10 ml). The combined organic layers were washed withsaturated saline (30 ml). After the organic layer was dried (MgSO₄), thesolvent was distilled off under reduced pressure, and then the obtainedresidue was chromatographed on silica gel and recrystallized fromethanol-ethyl acetate-ethyl ether to yield white crystalline powders(0.12 g, 95%).

Example 5-2 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2,6-difluorobenzyl)-2-[4-(2-hydroxyacetylamino)phenyl]-5-isobutyryl-4-oxothieno[2,3-b]pyridinehydrochloride

The white crystalline powders obtained in Example 5-1 were dissolved indichloromethane (1.0 ml), and then to this solution was added anethanolic solution containing 10 N hydrogen chloride (1.0 ml) with icecooling. The solvent was distilled off under reduced pressure, and thenthe residue was recrystallized from ethanol-ethyl acetate to yield whitecrystalline powders (0.12 g, 84%).

mp 197-199° C.

Elemental analysis for C₃₅H₃₃N₃O₄SF₂ HCl 2.5H₂O C (%) H (%) N (%)

Calculated: 59.11; 5.53; 5.91.

Found: 59.06; 5.25; 5.93.

¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H, d), 2.10(3H, s), 3.63(2H,s),3.72(1H,br), 4.09-4.17(1H,m), 4.16(2H,s), 4.24(2H,s), 5.24(2H,s),6.99(2H,t), 7.18-7.23(5H,m), 7.35-7.45(1H,m), 7.63(2H,d), 7.73(2H,d),8.25(1H,s), 8.59(1H,s).

Example 6

Using the compounds obtained in Example 4-2 and Example 4-3, thefollowing compounds (Examples 6-1 to 6-2) were obtained in the samemanner as in Example 5. Example 6-1

Yield: 76%

mp 192-194° C.

Example 6-2

Yield: 81%

mp 193-195° C.

Example 7 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2,6-difluorobenzyl)-2-(4-isobutyrylaminophenyl)-5-(2-hydroxy-2-methylpropionyl)-4-oxothieno[2,3-b]pyridine

The brown amorphous crystal (0.13 g) obtained by repeating the methoddescribed in Reference Example 33 using the compound obtained inReference Example 31-10 was hydrolyzed with potassium actetate by thesame method as described in Reference Example 34. The solid obtained waschromotographed on silic gel to romove the compound obtained inReference Example 34, and the byproduct was recrystallized from methanolto yield colorless crystalline powders (0.03 g).

mp 170-172° C.

Elemental analysis for C₃₇H₃₇N₃O₄SF₂ 1/6H₂O C (%) H (%) N (%)

Calculated: 67.26; 5.69; 6.36.

Found: 67.18; 5.74; 6.46.

¹H-NMR (300 MHz, CDCl₃) δ: 1.28(6H,d), 1.49(6H,s), 2.06(3H,s),2.51-2.60(1H,m), 3.61(2H,s), 4.13(2H,s), 5.31(2H,s), 7,01(2H,t),7.12-7.21(5H,m), 7.37-7.47(2H,m), 7.64(2H,d), 7.74(2H,d), 8.04(1H,s),8.35(1H,s).

IR (KBr): 3466, 1669, 1499, 1313, 1075 cm⁻¹.

Example 8 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2,6-difluorobenzyl)-2-(4-isobutyrylaminophenyl)-5-(3-hydroxy-2-methylpropionyl)-4-oxothieno[2,3-b]pyridine

The compound obtained in Reference Example 34 (0.56 g) was dissolved intrifluoroacetic acid (10 ml) and stirred at room temperature for 1 hour.The reaction mixture was concentrated to dryness under reduced pressure,and the obtained residue was partitioned between dichloromethane (30 ml)and saturated aqueous sodium bicarbonate (60 ml), and then the waterlayer was again extracted with dichloromethane (10 ml). The combinedorganic layers were washed with saturated saline (30 ml). After theorganic layer was dried (MgSO₄), the solvent was distilled of f underreduced pressure to yield brown amorphous crystals (0.42 g), which waschromatographed on silica gel to yield yellow crystals (0.07 g, overallrecovery rate as calculated from the compound obtained in ReferenceExample 31-10 12%).

¹H-NMR (300 MHz, CDCl₃) δ: 1.15 (3H, d), 1.26(6H, d), 2.07(3H, s),2.53-2.62(1H, m), 3.58(2H, d), 3.66 (2H, d), 3.82(1H, dd), 3.93(1H, dd),4.03(1H, d), 4.22(1H, d), 4.27-4.37(1H, m), 5.26(2H, s), 6.99(2H, t),7.13-7.21(5H, m), 7.36-7.46(1H,m), 7.63(2H, d), 7.67(2H, d), 7.79(1H,s), 8.30(1H, s).

Example 9 Production of(R)-4,7-dihydro-2-[4-(3-t-butoxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

To a solution of the compound obtained in Reference Example 29 (1.14 g,2.0 mmol) in dichloromethane (20 ml) were added diisopropylethylamine(520 mg, 4 mmol) and (2R)-3-t-butoxy-2-methylpropanoic acid (0.43 g, 3mmol), followed by stirring with ice cooling. To this solution was addedbenzotriazol-1-yloxytrisdimethylaminophosphonium hexafluorophosphate(BOP reagent) (1.33 g, 3 mmol). After stirring with ice cooling for 1hour, the solution was further stirred at room temperature for 4 hours.The reaction mixture was concentrated to dryness under reduced pressure,and then the obtained residue was partitioned between water (50 ml) andchloroform (50 ml). The water layer was again extracted with chloroform(10 ml). The combined extracts were washed with saline, and dried(MgSO₄), the solvent was distilled off under reduced pressure. Theobtained residue was chromatographed on silica gel to yield yellowpowders (1.11 g, 78%).

¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H,d), 1.25(3H,d), 1.30(9H,s),2.62(3H,s), 2.63-2.71(1H,m), 3.51(1H,t like), 3.61(1H,dd), 3.64(2H,s),4.18(2H,s), 4.18(1H,quint), 5.27(2H,s), 7.00(2H,t), 7.14-7.22(5H,m),7.36-7.46(1H,m), 7.60(2H,d), 7.76(2H,d), 8.28(1H, s), 9.00(1H,s).

Example 10 Example 10-1 Production of(R)-4,7-dihydro-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

The compound obtained in Example 9 (11.11 g) was dissolvedtrifluoroacetic acid (5 ml) under ice cooling conditions, and then afterstirring for 1 hour, the solution was allowed to warm to roomtemperature and stirred for further 12 hours. The reaction mixture wasconcentrated to dryness under reduced pressure, and then the residue wasdissolved in methanol (20 ml) and again cooled with ice, and then a 5 Nsolution of sodium hydroxide was added to reach pH 9.0. The reactionmixture was stirred for 30 minutes and further stirred at roomtemperature for 30 minutes. The reaction mixture was concentrated todryness under reduced pressure at low temperature, and then the obtainedresidue was chromatographed on silica gel. The light yellow amorphouscrystals obtained (0.83 g, 82%) were recrystallized from ether to yieldlight yellow powdery crystals (0.69 g).

mp 161-162° C.

Elemental analysis for C₃₇H₃₇N₃O₄SF₂ 0.5H₂O C (%) H (%) N (%)

Calculated: 66.65; 5.74; 6.30.

Found: 66.47; 5.73; 6.10.

¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H,d), 1.28(3H,d), 2.11(3H,s),2.71(1H,m), 3.65(2H,s), 3.83-3.85(2H,m), 4.16(1H,quint), 4.18(2H,s),5.27(2H,s), 7.00(2H,t), 7.14-7.23(5H,m), 7.36-7.44(1H,m), 7.64(2H,d),7.77(2H,d), 8.09(1H, s), 8.28(1H,s).

FAB-Mass m/z 658(MH)⁺

Example 10-2 Production of(R)-4,7-dihydro-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridinehydrochloride

The title compound was obtained in the same manner as in Example 1.

mp 214-216° C.

Example 11 Example 11-1 Production of4,7-dihydro-2-[4-(2-hydroxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

To a solution of the compound obtained in Reference Example 29 (1.14 g,2.0 mmol) in dichloromethane (30 ml) were added diisopropylethylamine(1.04 g, 8 mmol) and 2-hydroxyisobutanoic acid (0.416 g, 4 mmol),followed by stirring under ice cooling conditions. To this solution wasadded benzotriazol-1-yloxytrisdimethylaminophosphoniumhexafluorophosphate (BOP reagent) (1.76 g, 4 mmol). After stirring underice cooling conditions for 1 hour, the solution was further stirred atroom temperature for 4 days. The reaction mixture was concentrated todryness under reduced pressure, and then the obtained residue waspartitioned between water (50 ml) and chloroform (50 ml). The waterlayer was again extracted with chloroform (10 ml). The combined extractswere washed with saline, and dried (MgSO₄), the solvent was distilledoff under reduced pressure. The obtained residue was chromatographed onsilica gel and recrystallized from ether to yield yellow powderycrystald (0.56 g, 43%).

mp 178-180° C.

Elemental analysis for C₃₇H₃₇N₃O₄SF₂ 0.5H₂O C (%) H (%) N (%)

Calculated: 66.65; 5.74; 6.30.

Found: 66.54; 5.49; 6.36.

¹H-NMR (300 MHz, CDCl ₃) δ: 1.18(6H,d), 1.58(6H,s), 2.10(3H,s),3.05(1,brs), 3.64(2H,s), 4.16(2H,s), 4.12-4.22(1H,m), 5.23(2H,s),6.99(2H,t), 7.05-7.25(5H,m), 7.34-7.44(1H,m), 7.64(2H,d), 7.75(2H,d),8.25(1H, s), 8.86(1H, s).

Example 11-2 Production of4,7-dihydro-2-[4-(2-hydroxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridinehydrochloride

The title compound was obtained in the same manner as in Example 1.

mp 213-215° C.

Example 12 Example 12-1 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridine

To a solution of the compound obtained in Reference Example 29 (0.57 g,1.0 mmol) in dichloromethane (10 ml) were added diisopropylethylamine(0.52 g, 4 mmol) and 2-hydroxycyclopropanecarboxylic acid (0.204 g, 2mmol), followed by stirring under ice cooling conditions. To thissolution was added benzotriazol-1-yloxytrisdimethylaminophosphoniumhexafluorophosphate (BOP reagent) (1.76 g, 4 mmol). After stirring underice cooling conditions for 1 hour, the solution was further stirred atroom temperature for 4 days. The reaction mixture was concentrated todryness under reduced pressure, and then the obtained residue waspartitioned between water (50 ml) and chloroform (50 ml). The waterlayer was again extracted with chloroform (10 ml). The combined extractswere washed with saline, and dried (MgSO₄), the solvent was distilledoff under reduced pressure. The obtained residue was chromatographed onsilica gel and recrystallized from ether to yield yellow powderycrystals (0.27 g, 41%).

¹H-NMR (300 MHz, CDCl₃) δ: 1.16-1.20(2H,m), 1.18(6H, d),1.48-1.51(2H,m), 2.09(3H,s), 3.64(2H,s), 3.95(1H,br s), 4.14(2H,s),4.12-4.19(1H,m), 5.20(2H,s), 6.99(2H,t), 7.10-7.25(5H,m),7.34-7.46(1H,m), 7.57(2H,d), 7.70(2H,d), 8.21(1H, s), 8.82(1H, s).

Example 12-2 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridinehydrochloride

The title compound, white powdery crystals (0.12 g) was obtained in thesame manner as in Example 1. Solvent for recrystallization:ethanol-ether

mp 220-222° C.

Elemental analysis for C₃₇H₃₅N₃O₄SF₂ HCl 0.5H₂O C (%) H (%) N (%)

Calculated: 63.37; 5.32; 5.99.

Found: 63.35; 5.24; 5.82.

Example 13 Example 13-1 Production of4,7-dihydro-2-[4-(3-hydroxy-3-methylbutyrylamino)phenyl]-7-(2,6-difluarobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

To a solution of the compound obtained in Reference Example 29 (1.14 g,2.0 mmol) in dichloromethane (10 ml) were added diisopropylethylamine(1.04 g, 8 mmol) and 3-hydroxy-3-methylbutanoic acid (0.47 g, 4 mmol),followed by stirring under ice cooling conditions. To this solution wasadded benzotriazol-1-yloxytrisdimethylaminophosphoniumhexafluorophosphate (BOP reagent) (1.76 g, 4 mmol). After stirring underice cooling conditions for 1 hour, the solution was further stirred atroom temperature for 4 days. The reaction mixture was concentrated todryness under reduced pressure, and then the obtained residue waspartitioned between water (50 ml) and chloroform (50 ml). The waterlayer was again extracted with chloroform (10 ml). The combined extractswere washed with saline, and dried (MgSO₄), the solvent was distilledoff under reduced pressure. The obtained residue was chromatographed onsilica gel and recrystallized from ether to yield yellow powderycrystals (0.50 g, 37%).

¹H-NMR (300 MHz, CDCl₃) δ: 1.17(6H,d), 1.39(6H,s), 2.11(3H,s),2.58(2H,s), 3.12(1H, br s), 3.65(2H,s), 4.12-4.19(1H,m), 4.18(2H,s),5.27(2H,s), 7.00(2H,t), 7.10-7.23(5H,m), 7.32-7.44(1H,m), 7.61(2H,d),7.79(2H,d), 8.24(1H, s), 8.28(1H, s).

Example 13-2 Production of4,7-dihydro-2-[4-(3-hydroxy-3-methylbutyrylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridinehydrochloride

The title compound, white powdery crystals (0.42 g) were obtained in thesame manner as in Example 1. Solvent for recrystallization: ethanol

mp 216-218° C.

Elemental analysis for C₃₈H₃₉N₃O₄SF₂ HCl H₂O C (%) H (%) N (%)

Calculated: 62.84; 5.83; 5.79.

Found: 62.70; 5.75; 5.82.

Example 14

The following compounds (Examples 14-1 through 14-10) were produced bythe same method as described in Examples 9, 11, 12, and 13, using thecompound obtained in Reference Example 29, and also using variouscarboxylic acid compounds in place of, for example,(2R)-3-t-butoxy-2-methylpropanoic acid, which was used in Example 9.

Example 14-1

Yield: 46%

Amorphous.

¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H,d), 1.25(3H,d), 1.30(9H,s),2.11(3H,s), 2.60(1H,br), 3.51(1H, t like), 3.59-3.65(1H,m), 3.65(2H,s),4.17(1H,q), 4.18(2H,s), 5.27(2H,s), 7.00(2H, t), 7.12-7.22(5H,m),7.38-7.44(1H,m), 7.60(2H,d), 7.75(2H,d), 8.28(1H, s), 9.00(1H, s).

Example 14-2

Yield: 47%

mp 145-150° C.

Example 14-3

Yield: 99%

mp 96-98° C.

Example 14-4

Yield: 57%

mp 92-94° C.

Example 14-5

Yield: 71%

mp 57-62° C.

Example 14-6

Yield: 92%

mp 79-81° C.

Example 14-7

Yield: 97%

mp 91-96° C.

Example 14-8

Yield: 91%

mp 75-80° C.

Example 14-9

Yield: 87%

mp 83-88° C.

Example 14-10

Yield: 50%

mp 130-135° C.

Example 15

Using the compound obtained in Example 14, the following compounds(Examples 15-1 to 15-6) in the same manner as in Example 10.

Example 15-1

Yield: 69%

mp 161-162° C.

Example 15-2

Yield: 94%

mp 153-155° C.

Example 15-3

Yield: 62%

mp 224-226° C.

Example 15-4

Yield: 56%

mp 230-232° C.

Example 15-5

Yield: 90%

mp 92-94° C.

Example 15-6

Yield: 34%

mp 107-112° C.

Example 16 Example 16-1 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-N′-methoxyureidophenyl)-4-oxothieno[2,3-b]pyridine

To a solution of the compound obtained in Reference Example 29 (0.40 g,0.7 mmol) in dichloromethane (15 ml) were added triethylamine (0.20 ml,1.43 mmol) and N,N′-carbonyldiimidazole (0.228 g, 1.41 mmol), followedby stirring under ice cooling conditions. After the solution was allowedto warm to room temperature and further stirred for 2.5 days, to themixture were added O-mehtylhydroxylamine hydrochloride (0.59 g, 7.06mmol) and triethylamine (0.98 ml, 7.03 mmol) with ice cooling. While thesolution was allowed to return to room temperature, it was stirred for1.5 hours. After dilution with water (50 ml), the reaction mixture wasextracted with chloroform (10 ml). The water layer was again extractedwith chloroform (10 ml). The combined extracts were washed with saline,and dried (MgSO₄), the solvent was distilled off under reduced pressure.The obtained residue was chromatographed on silica gel to yield yellowamorphous powders (0.349 g, 77%).

¹H-NMR (300 MHz, CDCl₃) δ: 1.19(6H,d), 2.17(3H,s), 3.72(2H,brs),3.83(3H,s), 4.10-4.20(1H,m), 4.22(2H,brs), 5.28(2H,s), 7.00(2H,t),7.15-7.24(5H,m), 7.37-7.46(2H,m), 7.59(2H,d), 7.75(2H,d), 7.83(1H, br),8.31(1H,s).

Example 16-2 Production of4,7-dihydro-2-(4-N′-methoxyureidophenyl)-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridinehydrochloride

The title compound, white amorphous powders (0.335 g) were obtained inthe same manner as in Example 1. Solvent for recrystallization: ethylacetate-ether

mp 225-230° C.

Elemental analysis for C₃₅H₃₄N₄O₄SF₂ HCl 0.5H₂O C (%) H (%) N (%)

Calculated: 60.90; 5.26; 8.12.

Found: 60.80; 5.07; 8,17.

Example 17 Example 17-1 Production of(R)-4,7-dihydro-2-[4-(2,3-dihydroxypropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

To a 2 N aqueous solution of potassium hydroxide was added(R)-(+)-2,2-dimethyl-1,3-dioxolan-4-carboxylic acid methyl ester (1.00g, 6.24 mmol), followed by stirring under ice cooling conditions for 3hours. After neutralization with hydrochloric acid, the solution wassalted out and extracted with ethyl acetate (50 ml) to yield(R)-(+)-2,2-dimethyl-1,3-dioxolan-4-carboxylic acid as colorlessamorphous powders (0.72 g). To a solution of the compound obtained inReference Example 29 (0.572 g, 1.0 mmol) in dichloromethane (5 ml) wereadded diisopropylethylamine (1.04 g, 8 mmol) and the(R)-(+)-2,2-dimethyl-1,3-dioxo-lane-4-carboxylic acid obtained (0.292 g,2 mmol), followed by stirring under ice cooling conditions. To thissolution was added benzotriazol-1-yloxytrisdimethylaminophosphoniumhexafluorophosphate (BOP reagent) (0.885 g, 2 mmol). After stirringunder ice cooling conditions for 1 hour, the solution was furtherstirred at room temperature for 4 days. The reaction mixture wasconcentrated to dryness under reduced pressure, and then the obtainedresidue was partitioned between water (50 ml) and chloroform (50 ml).The water layer was again extracted with chloroform (10 ml). Thecombined extracts were washed with saline, and dried (MgSO₄), thesolvent was distilled off under reduced pressure. The obtained residuewas chromatographed on silica gel to yield colorless amorphous powders(0.5 g),which was then dissolved in trifluoroacetic acid (10 ml) andstirred at room temperature for 16 hours. The reaction mixture wasconcentrated to dryness under reduced pressure and treated with alkali,after which it was partitioned between chloroform (50 ml) and water (50ml), and then the water layer was again extracted with chloroform (10ml). The combined extracts were washed with saline, and dried (MgSO₄),the solvent was distilled off under reduced pressure. The obtainedresidue was chromatographed on silica gel to yield 0.5 g of colorlessamorphous powders (76%).

¹H-NMR (300 MHz, DMSO-d₆) δ: 1.09(6H,d), 1.95(3H,s), 3.51(2H,br),3.55-3.72(2H,m), 4.01-4.08(4H,m), 4.84(1H,t), 5.63(2H,brs), 5.80(1H,d),7.15-7.24(7H,m), 7.49-7.57(1H,m), 7.66(2H,d), 7.84(2H,d), 8.53(1H,s),9.82(1H,s).

Example 17-2 Production of(R)-4,7-dihydro-2-[4-(2,3-dihydroxypropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridinehydrochloride

The title compound, white powdery crystals (0.43 g) were obtained in thesame manner as in Example 1. Solvent for recrystallization:ether-ethanol

mp 146-148° C.

Example 18 Production of4,7-dihydro-2-(4-N′-hydroxy-N′-methoxyureidophenyl)-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

To a solution of the compound obtained in Reference Example 29 (1.0 g,1.75 mmol) in dichloromethane (40 ml) were added and triethylamine (0.49ml, 3.50 mmol) and N,N′-carbonyldiimidazole (0.567 mg, 3.50 mmol),followed by stirring under ice cooling conditions. After the solutionwas allowed to warm to room temperature and further stirred for 17hours, to the mixture was added N-methylhydroxylammonium chloride (730mg, 8.75 mmol) and triethylamine (1.23 ml, 8.75 mmol) with ice cooling.While the solution was allowed to warm to room temperature, it wasstirred for 5 hours. The reaction mixture was partitioned betweenchloroform (300 ml) and aqueous sodium bicarbonate (saturated, 200 ml),and then the organic layer was again washed with saline (200 ml). Afterthe organic layer was dried (MgSO₄), the solvent was distilled off underreduced pressure. The residue obtained was chromatographed on silica gelto yield pale yellow amorphous powder (0.38 g, 34%).

¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H, d), 2.15(3H, s), 3.26(3H, s),3.71(2H, s), 4.19(2H, s), 5.25(2H, s), 7.00(2H, t), 7.22-7.26(5H, m),7,41(1H, m), 7.49(2H,d), 7.60(2H,d), 8.06(1H, s), 8.26(1H, s).

Elemental analysis for C₃₅H₃₄N₄O₄SF₂ 0.8H₂O C (%) H (%) N (%)

Calculated: 63.78; 5.44; 8.50.

Found: 63.73; 5.24; 8.41.

Example 19 Production of2-[4-[(1-acetoxycyclopropyl)carbonylamino]phenyl]-3-(N-benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-4-oxothieno[2,3-b]pyridine

To a solution of the compound obtained in Reference Example 28 (0.42 g,0.7 mmol) and triethylamine (0.08 g, 0.8 mmol) in dichloromethane (10ml) was added 1-acetoxycyclopropanecarbonyl chloride (0.13 g, 0.8 mmol)with ice-cooling. The mixture was stirred at 0° C. for 30 minutes and atroom temperature for 30 minutes. The reaction mixture was diluted withchloroform (20 ml) and the solution was washed with aqueous sodiumbicarbonate (saturated, 20 ml), and brine (20 ml). After the organiclayer was dried (Na₂SO₄), the solvent was distilled off under reducedpressure. The residue obtained was chromatographed on silica gel toyield pale yellow amorphous powders (0.34 g, 66%).

¹H-NMR (300 MHz, CDCl₃) δ: 1.22(2H, dd), 1.68(2H, dd), 2.12(3H, s),2.21(3H, s), 3.64(2H, s), 4.13(2H, s), 5.28(2H, s), 7.01(2H, t),7.17-7.23(5H, m), 7.37-7.44(3H, m), 7.51-7.56(1H, m), 7.60(2H, d),7.81(2H, d), 7.87-7.90(3H, m), 7.94(1H, S).

FAB-Mass m/z 732 (MH)⁺

Example 20

Using the compound obtained in Reference Example 28, the followingcompound Examples 20-1 was obtained in the same manner as in Example 19

Example 20-1

Yield: 80%

¹H-NMR (300 MHz, CDCl₃) δ: 1.77(6H, s), 2.12(3H, s), 2.17(3H, s),3.63(2H, s), 4.13(2H, s), 5.29(2H, s), 7.01(2H, t), 7.15-7.23(5H, m),7.38-7.45(3H, m), 7.51-7.56(1H, m), 7.64(2H, d), 7.85(2H, d),7.88-7.90(3H, m), 7.94(1H, s).

FAB-Mass m/z 734(MH)⁺

Using the compound obtained in Reference Example 28, the followingcompound Examples 20-2 was obtained in the same manner as in Example 19

Example 20-2

Yield: 84%

¹H-NMR (300 MHz, CDCl₃) δ: 1.25(3H, d), 1.30(9H, s), 2.10(3H, s),2.65-2.67(1H, br m), 3.51(2H, t), 3.60(2H, s), 4.12(2H, s), 5.28(2H, s),7.00(2H, t), 7.16-7.23(5H, m), 7.37-7.42 (3H, m), 7.50-7.53(1H, m),7.61(2H, d), 7.80(2H, d), 7.89(2H, d), 7.94(1H, s), 9.01(1H, s).

FAB-Mass m/z 748(MH)⁺

Example 21 Production of3-(N-benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridine

A mixture of the compound obtained in Example 19 (0.24 g, 0.33 mmol) and5N sodium hydroxide (0.07 ml) in ethanol (8 ml) was stirred at roomtemperature for 1 hour. After the solvent was distilled of f underreduced pressure, the residue obtained was dissolved in chloroform (30ml) and the solution was washed with aqueous sodium bicarbonate(saturated, 20 ml) and brine (20 ml). After the organic layer was dried(Na₂SO₄), the solvent was distilled off under reduced pressure and theresidue obtained was recrystallized from chloroform-ether to yield paleyellow powders (0.17 g, 75%).

mp 186-188° C.

¹H-NMR (300 MHz, CDCl₃) δ: 1.15(2H, dd), 1.44(2H, dd), 2.05(3H, s),3.56(2H, s), 4.01(2H, s), 4.56(1H, br s), 5.24(2H, s), 7.00(2H, t),7.14-7.22(5H, m), 7.41(3H, t), 7.55(2H, d), 7.55(1H, s), 7.70(2H,d),7.88(2H, d), 7.90(1H, S), 8.88(1H, s).

Elemental analysis for C₄₀H₃₃N₃O₄SF₂1.0H₂O C (%) H (%) N (%)

Calculated: 67.88; 4.98; 5.94.

Found: 67.75; 4.70; 5.90.

FAB-Mass m/z 690 (MH)⁺

Example 22 Production of3-(N-benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-(2-hydroxy-2-methylpropionylamino)phenyl]-4-oxothieno[2,3-b]pyridine

Using the compound obtained in Example 20-1, the title compound wasobtained in the same manner as in Example 21. Solvent forrecrystallization: chloroform-ether

mp 222-223° C.

¹H-NMR (300 MHz, CDCl₃) δ: 1.57(6H, s), 2.09(3H, s), 2.50(1H, s),3.59(2H, s), 4.10(2H, s), 5.28(2H, s), 7.01(2H, t), 7.16-7.25(5H, m),7.38-7.44(3H, m), 7.51-7.56(1H, m), 7.67(2H, d), 7.82(2H, d), 7.89(2H,d), 7.93(1H, s), 8.82(1H, s).

FAB-Mass m/z 692 (MH)⁺

Example 23 Production of(R)-3-(N-benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-[(3-hydroxy-2-methylbutyrylamino)phenyl]-4-oxothieno[2,3-b]pyridine

Using the compound obtained in Example 20-2, the title compound wasobtained in the same manner as in Example 10. Solvent forrecrystallization: chloroform-ether

mp 145-146° C.

¹H-NMR (300 MHz, CDCl₃) δ: 1.25(3H, d), 2.07(3H, s), 2.63-2.70 (1H, m),2.84(1H, br s), 3.57(2H, s), 3.78-3.81(2H, m), 4.08(2H, s), 5.27(2H, s),7.00(2H, t), 7.17-7.20(5H, m), 7.37-7.43(3H, m), 7.51-7.56(1H, m),7.61(2H, d), 7.77(2H,d), 7.89(2H, d), 7.94(1H, s), 8.31(1H, br s).

FAB-Mass m/z 692 (MH)⁺

Preparation Example 1

Using 100 mg of the compound produced in Example 1-2, 165 mg of lactose,25 mg of corn starch, 4 mg of polyvinyl alcohol and 1 mg of magnesiumstearate, tablets are produced by a conventional method.

Preparation Example 2

The compound produced in Example 1-2 (5 g) is dissolved in distilledwater for injection to make a total volume of 100 ml. This solution isaseptically filtered through a 0.22 μm membrane filter (produced bySumitomo Electric Industries, Ltd. or Sartorius) and dispensed at 2 mlper washed sterile vial, followed by freeze-drying by a conventionalmethod, to yield a 100 mg/vial freeze-dried injectable preparation.

Preparation Example 3

Using 100 mg of the compound produced in Example 3, 165 mg of lactose,25 mg of corn starch, 4 mg of polyvinyl alcohol and 1 mg of magnesiumstearate, tablets are produced by a conventional method.

Preparation Example 4

The compound produced in Example 3 (5 g) is dissolved in distilled waterfor injection to make a total volume of 100 ml. This solution isaseptically filtered through a 0.22 μm membrane filter (produced bySumitomo Electric Industries, Ltd. or Sartorius) and dispensed at 2 mlper washed sterile vial, followed by freeze-drying by a conventionalmethod, to yield a 100 mg/vial freeze-dried injectable preparation.

Preparation Example 5

(1) Compound produced in Example 1-2  5 g (2) Lactose/crystallinecellulose (particles) 330 g (3) D-mannitol  29 g (4) Low-substitutionalhydroxypropyl cellulose  20 g (5) Talc  25 g (6) Hydroxypropyl cellulose 50 g (7) Aspartame  3 g (8) Dipotassium glycyrrhizinate  3 g (9)Hydroxypropylmethyl cellulose 2910  30 g (10) Titanium oxide  3.5 g (11)Yellow iron sesquioxide  0.5 g (12) Light silicic anhydride  1 g

Components (1), (3), (4), (5), (6), (7) and (8) are suspended ordissolved in purified water and coated on the core particles (2) toyield base fine subtilae, which are then further coated with components(9) through (11) to yield coated fine subtilae, which are then mixedwith component (12) to yield 500 g of 1% fine subtilae of the compound.These subtilae are divided to 500 mg folded subtilae.

Experimental Example 1

Preparation of ¹²⁵I-leuprorelin

To a tube containing 10 μl of a 3×10⁻⁴ M aqueous solution of leuprorelinand 10 μl of 0.01 mg/ml lactoperoxidase, 10 μl (37 MBq) of a solution ofNa¹²⁵I was added. After stirring, 10 μl of 0.001% H₂O₂ was added, and areaction was carried out at room temperature for 20 minutes. By adding700 μl of a 0.059% TFA (trifluoroacetic acid) solution, the reaction wasstopped, followed by purification by reversed-phase HPLC. The HPLCconditions used are shown below. ¹²⁵I-leuprorelin was eluted at aretention time of 26 to 27 minutes.

Column: TSKgel ODS-80™ (TM indicates a registered trademark; the sameapplies below) CTR (4.6 mm×10 cm)

Eluents:

Solvent A (0.05% TFA)

Solvent B (40% CH₃CN-0.05% TFA)

0 minute (100% Solvent A)—3 minutes (100% Solvent A)—7 minutes (50%Solvent A+50% Solvent B)—40 minutes (100% Solvent B)

Eluting temperature: Room temperature

Elution rate: 1 ml/min

Experimental Example 2

Preparation of a rat pituitary anterior lobe membrane fractioncontaining GnRH receptors

Anterior lobes of the pituitary glands were isolated from forty Wistarrats (8 weeks old, male), and washed with ice-cooled homogenate buffer[25 mM Tris (tris(hydroxymethyl)aminomethane)-HCl, 0.3 M sucrose, 1 mMEGTA (glycol-etherdiamine-N,N,N′,N′-tetraacetic acid), 0.25 mM PMSF(phenylmethylsulfonyl fluoride), 10 U/ml aprotinin, 1 mg/ml pepstatin,20 mg/ml leupeptin, 100 mg/ml phosphoramidon, 0.03% sodium azide, pH7.5]. The pituitary tissue was floated in 2 ml of the homogenate bufferand homogenized using a Polytron homogenizer. The homogenate wascentrifuged at 700×g for 15 minutes. The supernatant was taken in anultracentrifuge tube and centrifuged at 100,000×g for 1 hour to providea membrane fraction pellet. This pellet was suspended in 2 ml of assaybuffer [25 mM Tris-HCl, 1 mM EDTA (ethylenediaminetetraacetic acid), 0.1BSA (bovine serum albumin), 0.25 mM PMSF, 1 mg/ml pepstatin, 20 mg/mlleupeptin, 100 mg/ml phosphoramidon, 0.03% sodium azide, pH 7.5) and thesuspension was centrifuged at 100,000×g for 1 hour. The membranefraction recovered as a pellet was resuspended in 10 ml of assay buffer,divided into portions, preserved at −80° C. and thawed when needed.

Experimental Example 3

Preparation of CHO (Chinese hamster ovarian) cell membrane fractioncontaining human GnRH receptor

Human GNRH receptor-expressing CHO cells (EP-A-678577) (10⁹ cells) weresuspended in phosphate-buffered saline supplemented with 5 mM EDTA(ethylenediaminetetraacetic acid) (PBS-EDTA) and centrifuged at 100×gfor 5 minutes. To the cell pellet, 10 ml of a cell homogenate buffer (10mM NaHCO₃, 5 mM EDTA, pH 7.5) was added, followed by homogenizationusing the Polytron homogenizer. After centrifugation at 400×g for 15minutes, the supernatant was transferred to an ultracentrifugation tubeand centrifuged at 100,000×g for 1 hour to yield a membrane fractionprecipitate. This precipitate was suspended in 2 ml of an assay bufferand centrifuged at 100,000×g for 1 hour. The membrane fraction recoveredas a precipitate was again suspended in 20 ml of the assay buffer,dispensed, and stored at −80° C. before use upon thawing.

Experimental Example 4

Determination of ¹²⁵I-leuprorelin binding inhibition rate

The rat and human membrane fractions prepared in Experimental Examples 2and 3 were diluted with the assay buffer to yield a 200 mg/ml dilution,which was then dispensed at 188 ml per tube. Where the rat pituitaryanterior lobe membrane fraction was used, to each tube, 2 ml of asolution of 0.1 mM compound in 60% DMSO (dimethyl sulfoxide) and 10 μlof 38 nM ¹²⁵I-leuprorelin were added simultaneously. Where the cellmembrane fraction of the CHO with human GnRH receptors expressed, toeach tube, 2 ml of a solution of 2 mM compound in 60% DMSO and 10 μl of38 nM ¹²⁵I-leuprorelin were added simultaneously. To determine maximumbinding quantity, a reaction mixture of 2 μl of 60% DMSO and 10 μl of 38nM ¹²⁵I-leuprorelin was prepared. To determine non-specific bindingamount, a reaction mixture of 2 μl of 100 μM leuprorelin in solution in60% DMSO and 10 μl of 38 nM ¹²⁵I-leuprorelin was prepared.

Where the rat or bovine pituitary anterior lobe membrane fraction wasused, the reaction was conducted at 4° C. for 90 minutes. Where themembrane fraction of the CHO with human GnRH receptors expressed wasused, the reaction was carried out at 25° C. for 60 minutes. After eachreaction, the reaction mixture was aspirated and filtered through apolyethyleneimine-treated Whatman glass filter (GF-F). After thisfiltration, the radioactivity of ¹²⁵I-leuprorelin remaining on thefilter paper was measured with a γ-counter. The expression(TB-SB)/(TB-NSB)×100 (where SB=radioactivity with the compound added,TB=maximum bound radioactivity, NSB=nonspecifically bound radioactivity)was calculated to find the binding inhibition rate (%) of each testcompound. Furthermore, the inhibition rate was determined by varying theconcentration of the test substance and the 50% inhibitory concentration(IC₅₀ value) of the compound was calculated from Hill plot. The resultsare shown in below.

binding inhibition rate (%) Rat Human IC₅₀ (μM) Test Compound (1 μM) (20μM) Rat Human Compd. of Ex. 12 96 102 0.06 0.0001 Compd. of Ex. 3 62 NT0.6  0.0002 NT: not measured

Experimental Example 5

Suppression of blood LH in castrated monkeys

The compound produced in Example 1-2 was orally administered tocastrated male cynomolgus monkeys (Macaca fascicularis), and blood LHwas quantified. The male cynomolgus monkeys, used at 3 years 8 months to7 years 7 months of age at time of experimentation, had been castratedmore than half a year prior to the examination. Test animals [n=2;compound (1) and compound (2)] were given 30 mg/kg (3 ml/kg) of thecompound suspended in 0.5% methyl cellulose at a final concentration of1% by oral administration, and control animals (n=3; control (1),control (2) and control (3)] were given 3 ml/kg of the 0.5% methylcellulose dispersant alone by oral administration. At 24 hours andimmediately before administration and at 2, 4, 6, 8, 24, and 48 hoursafter administration, blood was collected for heparinized plasma samplesvia the femoral vein and immediately stored under freezing conditions.

Plasma LH concentrations were determined by a bioassay using mousetesticular cells. The testicular cells were collected from male BALB/cmice (8 to 9 weeks of age) and washed three times with 1 ml ofDulbecco's modified Eagle medium (DMEM-H) containing 20 mM HEPES and0.2% BSA per testis. After incubation at 37° C. for 1 hour, the cellswere passed through a nylon mesh filter (70 μm) and dispensed to testtubes at 8×10⁵ cells /tube. After the cells were washed twice with 0.4ml of DMEM-H, 0.4 ml of a DMEM-H solution containing either equine LH(Sigma Company), as the standard LH, or monkey plasma, previouslydiluted up to 1,000 fold, as the test sample, was added, followed by areaction at 37° C. for 2 hours. The testosterone concentration in theculture supernatant was determined by a radioimmunoassay (CISDiagnostics Company), and the LH concentration in the test monkey plasmawas calculated from the standard curve for the standard equine LH.

The results are given together in FIG. 1.

The compound indicated is the compound obtained in Example 1-2.

For the control (1), the control (2), and the control (3), changes overtime in the lh concentrations in each animal are expressed as percentratios to the respective reference values, which comprise the LHconcentrations immediately before administration in each control animal(cynomolgus monkey). Similarly, for the compound (1) and the compound(cynomolgus monkey) receiving the compound of Example 1-2 are expressedas percent ratios to the respective reference values, which comprise thLH concentrations immediately before administration of the compound. Thetime of administration was defined as zero (0), and hours before andafter administration shown by minus and plus, respectively.

Industrial Applicability

The compound of present invention possesses excellencygonadotropin-releasing hormone antagonizing activity. It is also good inoral absorbability and excellent in stability and pharmacokinetics. Withlow toxicity, it is also excellent in safety.

The compound of the present invention can therefore be used as aprophylactic or therapeutic agent for hormone-dependent diseases etc.Specifically, it is effective as a prophylactic or therapeutic agent forsex hormone-dependent cancer, pituitary tumor), prostatic hypertrophy,hysteromyoma, endometriosis, precocious puberty, amenorrhea syndrome,multilocular ovary syndrome, pimples etc, or as a pregnancy regulator(e.g., contraceptive) infertility remedy or menstruation regulator, Itis also effective as an animal estrous regulator, food meat qualityimproving agent or animal growth regulator in the field of animalhusbandry, and as a fish spawning promoter in the field of fishery.

What is claimed is:
 1. A compound of the formula:

wherein R¹ represents a C₃₋₇ cycloalkyl group which may be substituted,a C₁₋₆ alkoxyamino group which may be substituted or a hydroxyaminogroup which may be substituted; and R² represents a C₁₋₇ alkyl groupwhich may be substituted or a phenyl group which may be substituted; ora salt thereof.
 2. A compound of claim 1 or a salt thereof, wherein R¹is (1) a C₃₋₇ cycloalkyl group which may be substituted by 1 to 3substituents selected from the group consisting of (i) hydroxy, (ii)C₁₋₇ acyloxy, (iii) amino which may be substituted by 1 or 2substituents selected from the group consisting of C₁₋₆ alkoxy-carbonyl,benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iv)C₁₋₁₀ alkoxy which may be substituted by 1 to 3 substituents selectedfrom the group consisting of C₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxyand (v) C₁₋₆ alkoxy-carbonyl, (2) a C₁₋₆ alkoxyamino group which may besubstituted by 1 to 5 substituents selected from the group consisting of(i) hydroxy, (ii) C₁₋₇ acyloxy, (iii) amino which may be substituted by1 or 2 substituents selected from the group consisting of C₁₋₆alkoxy-carbonyl, benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃ alkylsulfonyl andC₁₋₃ alkyl, (iv) C₁₋₁₀ alkoxy which may be substituted by 1 to 3substituents selected from the group consisting of C₃₋₇cycloalkyloxy-carbonyl and C₁₋₃ alkoxy and (v) C₁₋₆ alkoxy-carbonyl, or(3) a hydroxyamino group which may be substituted by 1 or 2 substituentsselected from the group consisting of (i) C₁₋₇ acyloxy, (ii) amino whichmay be substituted by 1 or 2 substituents selected from the groupconsisting of C₁₋₆ alkoxy-carbonyl, benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃alkylsulfonyl and C₁₋₃ alkyl, (iii) C₁₋₁₀ alkoxy which may besubstituted by 1 to 3 substituents selected from the group consisting ofC₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxy and (iv) C₁₋₆ alkyl; and R²is (1) a C₁₋₇ alkyl group which may be substituted by 1 to 5substituents selected from the group consisting of (i) hydroxy, (ii)C₁₋₇ acyloxy, (iii) amino which may be substituted by 1 or 2substituents selected from the group consisting of C₁₋₆ alkoxy-carbonyl,benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iv)C₁₋₁₀ alkoxy which may be substituted by 1 to 3 substituents selectedfrom the group consisting of C₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxyand (v) C₁₋₆ alkoxy-carbonyl, or (2) a phenyl group which may besubstituted by 1 to 5 substituents selected from the group consisting ofhalogen, C₁₋₃ alkyl and C₁₋₃ alkoxy.
 3. A compound of claim 1 or a saltthereof, wherein R¹ is a C₃₋₇ cycloalkyl group which may be substituted.4. A compound of claim 1 or a salt thereof, wherein R¹ is a C₃₋₇cycloalkyl group substituted by hydroxy.
 5. A compound of claim 1 or asalt thereof, wherein R¹ is a substituted C₃₋₇ cycloalkyl group.
 6. Acompound of claim 1 or a salt thereof, wherein R¹ is a cyclopropyl groupwhich may be substituted by hydroxy.
 7. A compound of claim 1 or a saltthereof, wherein R² is a branched C₃₋₇ alkyl group which may besubstituted.
 8. A compound of claim 1 or a salt thereof, wherein R² is aphenyl group which may be substituted.
 9. A compound of claim 1 or asalt thereof, wherein R² is a phenyl group.
 10. A compound of claim 1 ora salt thereof, wherein R¹ is a C₃₋₇ cycloalkyl group and R² is a C₁₋₆alkyl group.
 11. A compound of claim 1 or a salt thereof, wherein R¹ isa C₃₋₇ cycloalkyl group substituted by hydroxy, or a C₁₋₃ alkoxyaminogroup; and R² is an isopropyl group or a phenyl group.
 12. A compound ofclaim 1 or a salt thereof, wherein R¹ is a C₃₋₇ cycloalkyl group whichmay be substituted by a hydroxy or a C₁₋₃ alkyl-carbonyloxy, or a C₁₋₃alkoxyamino group; and R² is (1) an isopropyl group which may besubstituted by a hydroxy or (2) a phenyl group. 13.3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-cyclopropanecarbonylaminophenyl)-4-oxothieno[2,3-b]pyridineor a salt thereof. 14.3-(N-Benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridineor a salt thereof.
 15. A process for producing a compound of claim 1 ora salt thereof, which comprises reacting a compound of the formula:

wherein R² represents a C₁₋₇ alkyl group which may be substituted or aphenyl group which may be substituted, or a salt thereof, with i) acompound of the formula: R^(1a)COOH wherein R^(1a) represents a C₃₋₇cycloalkyl group which may be substituted, or a salt thereof or areactive derivative thereof; or ii) carbonyldiimidazole, phosgene or achloroformate, followed by reacting with a compound of the formula:R^(1b)H wherein R^(1b) represents a C₁₋₆ alkoxyamino group which may besubstituted or a hydroxyamino group which may be substituted, or a saltthereof.
 16. A process for producing a compound of claim 3 or a saltthereof, which comprises reacting a compound of the formula:

wherein R² represents a C₁₋₇ alkyl group which may be substituted or aphenyl group which may be substituted, or a salt thereof, with acompound of the formula: R^(1a)COOH wherein R^(1a) represents a C₃₋₇cycloalkyl group which may be substituted, or a salt thereof or areactive derivative thereof.
 17. A pharmaceutical composition whichcomprises a compound of claim 1 or a salt thereof and a pharmaceuticallyacceptable carrier or excipient.
 18. A method for antagonizinggonadotropin-releasing hormone in a mammal in need thereof, said methodcomprising administering, an effective amount of a pharmaceuticalcomposition of claim 17 to said mammal.
 19. A method for treating a sexhormone dependent disease in a mammal in need of such treatment, saidmethod comprising administering, an effective amount of a pharmaceuticalcomposition of claim 17 to said mammal.
 20. A method for antagonizinggonadotropin-releasing hormone in a mammal in need thereof whichcomprises administering to said mammal an effective amount of a compoundof claim 1 or a salt thereof.
 21. A method for manufacturing apharmaceutical composition, said method comprising, combining a compoundof claim 1 or a salt thereof with a pharmaceutically acceptable carrieror excipient. 22.3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-N′-methoxyureidophenyl)-4-oxothieno[2,3-b]pyridineor a salt thereof. 23.4,7-dihydro-2-(4-N′-methoxyureidophenyl)-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridinehydrochloride. 24.4,7-dihydro-2-(4-N′-hydroxy-N′-methoxyureidophenyl)-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridineor a salt thereof.